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NOT TO BE TAKEN FROM THIS ROOM

A STUDY OF AUTOTEIPLOIDS AND TRISOMICS

OF COMMON BARLEY, HORDSUM VULGARS L,

E. R, Kerber

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THE UNIVERSITY OF ALBERTA

A STUDY OF AUT0TRIPL0ID3 AND TRISOMIGS
OF COMMON BARLEY* HORDEUM VULGARE L.

A DISSERTATION

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE
OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF PLANT SCIENCE

Erich Rudolph Kerber

EDMONTON^ ALBERTA

APRIL* 1958

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ABSTRACT

Triploid plants of Hordeum vulgare L. were found in an F 2
intervarietal hybrid population derived from colchicine treated F]_ plants
and in a large nursery of the variety Gateway. Triploids occurred
spontaneously in Gateway with an estimated frequency of one in 6000 plants.
An attempt to produce triploids by crossing tetraploid with diploid
Gateway was unsuccessful.

At meiosis in the Gateway triploids univalents lying off the
equatorial plate during metaphase I were found to be distributed on
opposite sides of the plate at random. Furthermore, only univalents
located on the plate at the completion of metaphase I lagged and divided
equationally at anaphase I. At anaphase I the distribution of the extra
set of seven chromosomes to the poles was found to be in a binomial
frequency. Microcytes formed by diads located on the periphery of the
cells at anaphase I behaved as independent minute cells in succeeding
meiotic stages.

Among the progeny of the triploids no aneuploid plants
occurred with more than three extra chromosomes ; the majority were either
diploid or were primary trisomics. Data were given on the fertility and
on the transmission of the extra chromosome through the gametes of a
number of trisomics. Four morphologically distinct primary trisomic
types were described in Gateway. One of these was cytogenetically
demonstrated to be associated with Linkage Group II and independent of
the remaining known groups.

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ACKNOVJLED SEMEN TS

The writer expresses his thanks to the National Research
Council of Canada for financial assistance in the form of a
Studentship and Fellowship during the academic years 1954-55 and
1955-56, respectively; he is also grateful for the patient guidance
given by Dr* John Unrau throughout the course of the study. The
writer also expresses gratitude to his wife, Florence, for typing
the manuscript.

table: of contents

Page

INTRODUCTION . 1

PART I • TRIPIDIDS

REVIM OF LITERA1URE .•. 3

Origin and Occurrence of Triploids . 3

Morphology of Triploids ..... 7

Meiosis in Triploids . 7

First Division .. 7

Second Division . 18

Functional Gametes of Triploids . 19

MATERIALS AND METHODS . 21

OBSERVATIONS AND RESULTS . 22

Occurrence of Triploids .. 22

Experimental Production of Triploids . 23

Morphology of Triploids . 23

Meiosis in Triploids . 24

Metaphase I . 24

Behavior of Trivalents and Bivalents ... 26

Behavior of Univalents . 29

Anaphase I . 33

Telophase I .. 43

Interphase .. 44

Second Division ... 45

Viability of Pollen from Triploids .. 4S

Fertility of Triploids . 49

Progeny of Triploids . 52

TABLE OF CONTENTS (continued)

Page

DISCUSSION . 53

SUiviMARY .. 62

ERiijNOES .. 82

PART II : TRISOMICS

REVIEW OF LITERATURE . 64

MATERIALS AND METHODS . 68

OBSERVATIONS AND RESULTS . 69

Morphological Characteristics of Primary Trisomics •... 69

Fertility of Trisomics ... .. 72

Transmission of Trisomics . 74

Cytogenetic Identification of Gateway Trisomic T39 ... 76

DISCUSSION . 78

SUMMARY ... 80

REFERENCES . 86

A STUDY OF AU TOTRIPLOIDS AND TRISOMICS

OF COMMON ft a WJ.p.Y., iiORDLUk VULG-AkD L.

INTRODUCTION

Autotriploid plants possess three basic sets of homologous
chromosomes, whereas diploids have two. Triploids^ - have been reported
and studied in numerous genera. The investigations have dealt with their
natural occurrence and their experimental production, the pairing
relationships of the chromosomes at prophase, the behavior of trivalent
complexes and univalents at metaphase and anaphase of meiosis, and with
the chromosome numbers of functional gametes. Although these studies have
made valuable contributions to the elucidation of chromosome behavior
in general as well as to the cytogenetics of the species concerned, the
results have been varied and often inconclusive, particularly with regard
to the behavior of the extra set of chromosomes at meiosis.

Common barley, Hordeum vulgare L., is a diploid having 14
chromosomes. Reports on triploids of this species are relatively rare,
and the behavior of their chromosomes at meiosis has been described in
only one report. The usual experimental methods of obtaining triploids
in other species have been found generally unsuccessful in barley.

Among the aneuploid progeny produced by triploids, trisomics
(plants with one chromosome in triplicate) have been of particular value
in cytogenetic investigations. Since the genetic ratios for genes carried
by the extra chromosome are modified, trisomics are useful for associating
chromosomes with their respective linkage groups. This altered ratio

Hereafter the term ’triploid* refers to autotriploid.

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technique has been successfully applied’to Datura , tomato, corn and tobacco,
to mention a fevj plants. It is only recently that trisomies of barley
have been obtained and utilized in this manner.

According to published reports, four and possibly five of the
barley linkage groups have been identified -with their respective
chromosomes through the use of chromosome translocations and trisomies.

To date, however, a complete series of the seven possible primary barley
trisomies has not, apparently, been developed and made available to barley
cytogeneticists•

The discovery of a number of triploid plants in two field
populations of common barley prompted the present study, the first part
of which deals with the triploids and the second part with their trisomic
progeny.

The objectives in the first part of the study were 1) to
determine the frequency and origin of triploids that occurred spontaneously;
2) to attempt to produce triploids of barley experimentally; 3) to
contribute further to the knowledge of the behavior of chromosomes in
triploids, particularly of the extra set; and 4) to determine the frequency
of the various chromosomal types among the progeny of the triploids.

The objectives in the second part of the study were l) to
describe the morphological characteristics of a number of primary trisomies;
2) to determine the fertility of trisomies and the transmission of gametes
with an extra chromosome; and 3) to provide evidence for the association
of one trisomic type with its corresponding linkage group, which heretofore
had not been definitely shown to be independent of the remaining groups.

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PART I: TRIPL0ID3

REVIEW OF LITERATURE

Occurrence and Origin of Triploids

Triploid plants have been obtained from suitable experimental
crosses and from natural populations in which they occurred spontaneously.

Triploids have been produced experimentally by crossing an
autotetraploid with its related diploid in Datura (10, 12), Lolium
(339) > Lycopersicum (l6), Petunia (2l), Primula (17, 19), Secale (25),
and Zea (15, 47). In his extensive review Smith (56) cited no reports
on triploids of common barley obtained by this method. Tsuchiya (63),
however, produced a hypotriploid plant, 2n~ - 20, by crossing an
artificially induced autotetraploid with its related diploid of the
same variety. He further reported ( 64 ) a hybrid triploid obtained by
crossing the same autotetraploid with Hordeum spontaneum nigrum (2n z 14).
The latter is a wild species closely related to common cultivated barley.

The success with which triploids are obtained by intercrossing
autotetraploids with diploids depends on the species and the direction
in which the cross is made. Blakeslee et al. (10), and Buchholz and
Blakeslee (12) observed that in Datura, triploids were produced, from
the small proportion of viable seeds obtained, only when the tetraploid
was used as the female parent. The reciprocal cross was completely
incompatible due to pollen tube growth failure. A similar relationship
was found in Primula sinensis by Darlington (19).

^ Throughout this study the symbols x 2n' and ‘n‘ refer to the zygotic
and gametic chromosome complements, respectively.

When the autotetraploid of this species was pollinated by the diploid,
pollen tube growth appeared quite normal. In another study involving
the same cross Watkins (66) stated that fertilization failed to occur.
However, apparently fertilization must have occurred rarely since
Darlington obtained triploids from this cross. In the reciprocal
cross 2x^ pollen tubes were not functional in the style of 4* plants.

Randolph (43) found that in the cross 2x X 4x of Zea mays
about 98 per cent of the seed was abortive and less than 0.5 per cent
of the relatively well-filled seeds were viable, while the reciprocal
produced seed with a viability of less than five per cent. Cooper (15)
studied the development of the caryopsis of these reciprocal crosses
and concluded that the high degree of incompatibility was not due to non¬
fertilization but to failure of the caryopsis to reach a germinable stage;
endosperm development was abnormal and the embryo suffered from a lack
of nutrients. More normal development was noted in the 4x X 2x cross
than in the reciprocal. Triploid plants were obtained from seed of both
combinations.

According to Chin (14), when autotetraploid rye was pollinated
with the diploid, pollen tube growth was normal, and approximately 38
per cent of the florets set seed. The incompatibility of the reciprocal
cross was attributed to growth failure of the pollen tube. On the other
hand, Hakansson and Sllerstrom (25) found that in their stocks of rye,
fertilization occurred regularly in both combinations of the tetraploid
and diploid. However, only four triploid plants were obtained from 783

The symbol ’x‘ refers to the basic haploid chromosome number of a species.

tetraploid florets pollinated -with the diploid and the same number from
1275 diploid florets pollinated with the tetraploid. The almost complete
incompatibility in these matings was attributed to irregular development
and disintegration of the endosperm. Endosperm development was somewhat
more normal in the 4& X 2x cross, which probably accounted for the slightly
greater success of this combination.

Seed collapse following crosses between diploid and autotetraploid
races of Lycopersicum pimpinellifolium was studied by Cooper and Brink (16).
They concluded that incompatibility of 2x X 4x and 4x X 2x crosses was not
due to triploidy as such but to conditions surrounding the triploid
embryo within the seed. The occasional triploid obtained from the 4x X 2x
combination exhibited normal vegetative growth.

The spontaneous occurrence of triploids in diploid populations
has been noted in Canna (66), Lycopersicum (30, 50), Nicotiana (22),

Tulipa (42), Zea (33* 49), and in several genera of Qramineae » including
Avena, Hordeunij Saeala and Tr.iti.num ( 28 , 29, 36, 37). The relative
frequency in which they appeared has been determined in a few cases.

Lesley (30) found from one to 0.4 per cent triploids in two different
varieties of tomato. In the same species but different varieties Rick (50)
calculated a frequency of one triploid in about 1200 plants, or less than
2.08 per cent, and observed variation in frequency from season to season
within the same variety.

Triploids have occasionally occurred as members of twin seedlings.
Muntzing (37) found that of 2201 twin seedlings from 14 genera, including
Avena, Hordeum, Secale and Triticum , 2106 were diploid, while 95 had
deviating chromosome numbers of which 77 were triploid. From the data
of all species investigated there was noted a distinct tendency for one

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member of a heteroploid "twin to be triploid. The frequency of twins in
eight varieties of Hordeum vulgare was found to be about one in 5900
seedlings, and one twin member of 93 examined was triploid. Aase (2)
and Kostoff (28) have suggested that the frequency of twinning is
genetically controlled since it varies between species and between
varieties.

Several methods of origin have been proposed for the spontaneous
occurrence of triploids:

1. They have occurred through natural intercrossing of tetraploids
and diploids found within the species. The various aspects of this method
have been discussed above.

2. A commonly supposed origin is the union of an unreduced with
a reduced gamete of an otherwise normally behaving diploid plant.

Unreduced gametes may be the result of failure of one of the meiotic
divisions. 'That such unreduced gametes are formed has been rather
definitely demonstrated cytologically (2, 8 , 9* 35* 42* 66). Syndiploidy

' has been suggested as a further source of gametes with the diploid
chromosome number (20). This is the failure of separation of daughter
nuclei in divisions immediately preceding meiosis. It is thought that
fusion takes place after the pachytene stage of meiosis, since usually no
quadrivalents are produced. Aase (2) noted that pollen mother cells with
multiple euploidy were not infrequently found in routine studies of anther
material. It is logical to assume that similar phenomena occur to form
doubled egg cells. Puick (50) noted considerable variation from season to
season in the frequency of spontaneous occurrence of triploids in the same
variety of tomato and suggested that high temperatures during the growing

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season possibly influenced the production of diploid gametes. The fusion
of a reduced with an unreduced gamete has been inferred as the method of
origin of triploidy in Canna (5), Lycopersicum (30, 50), Tulipa (42),
and Zea (33, 46).

3. A third possible method suggested for the origin of triploids
is the fertilization of an egg cell by two male nuclei (10, 22).

4. The mode of origin of triploids from twin seedlings has
been attributed to embryonal development of a fertilized endosperm
cell or to a fertilized unreduced nucleus of a supernumary embryo sac.

(2). According to Muntzing (36), a supernumary macrospore mother cell
could give rise to an extra embryo sac having an unreduced chromosome
number, which when fertilized by haploid pollen would result in a triploid
zygote.

Morphology of Triploids

The morphological appearance of triploids varies from species
to species. Lesley (30) noted that the stems, leaves, and flowers of
triploid tomato were more or less gigantic, but the fruits were under¬
sized and few in number. Although triploid maize was observed to be
more vigorous than diploid, there was no striking morphological difference
(33). Lamm (29) found difficulty in distinguishing a triploid rye plant
from normal diploids, the former being only slightly more vigorous. The
triploid derived from crossing tetraploid common barley with the wild
species Hordeum spontaneum appeared to exhibit heterosis ( 64 ).

Meiosis in Triploids

First Division

According to the generally accepted hypothesis of pachytene

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pairing, homologous chromosomes synapse at random as pairing segments;
that is, only two chromosomes pair at one point (7, 20, 42, 58). Since
triploids possess three homologous chromosomes of each kind, equal
lengths of paired and unpaired chromosome segments should occur. The
variation in number of chiasmata formed in these paired lengths
determines the type of association observed at metaphase. If no
chiasmata are formed between two particular chromosomes in the paired
sectors, univalents occur. Studies of triploids of Kyacinthus (18, 20)
and Tulipa (42) have indicated that the frequency of chiasmata formation
is in proportion to the length of the chromosomes. Consequently, longer
chromosomes are more likely to form trivalents and, for the same reason,
more complex associations, while a relatively greater frequency of shorter
chromosomes will occur as univalents.

Myers (38) observed that at prophase of triploid Loleum perenne
there was an excess of paired and a deficiency of single chromosome strands.
However, never more than two chromosomes were associated at one point.

The excess of paired strands appeared to result from pairing of normally
nonhomologons segments. This form of illegitimate pairing evidently was
not accompanied by ehiasma formation, since only trivalents, bivalents,
and univalents occurred at metaphase in frequencies expected from normal
pairing.

Earlier papers on triploids of Canna (5), Datura (7), and
Hyacinthus (6) reported that only trivalents were regularly observed at
metaphase. However, more detailed later studies on these and other species
indicated that bivalents and univalents also occurred (13, 17, 21, 22, 29,
31, 33, 38, 39, 47, 63, 64, 65 ). Usually various combinations of

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trivalents, bivalents, and univalents were found at metaphase ; the
frequency of each varied from species to species and from cell to cell
within the same anther* The combined total of trivalents and bivalents
equalled the haploid chromosome number in true autotriploids ; pairing
rarely occurred between chromosomes of the haploid or extra set.

The five types of trivalent configurations that are
theoretically possible from normal synapais of three homologues (20)
are diagrammatically illustrated in Fig. 1. ‘The tandemr-chain and
tandem - V types require a minimum of two chiasmata, one in each arm,
while the triradial configuration also requires two chiasmata, both in

TRIFLE-

ARC

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Fig. 1. Pairing arrangements of three homologous chromosomes at diplotene
and resulting trivalent configurations at metaphase I.

DIPLOTENE METAPHASE

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the same arm; the ring-rod requires a minimum of three chiasmata, two
involving one arm and one the other, while two chiasmata in each arm
produce a triple-arc association.

In triploids of Datura and hyacinth Belling (7) found that
short chromosomes with median centromeres formed any one of the
trivalent configurations. Short chromosomes with subterminal centromeres,
such as occurred in hyacinth, formed more complicated configurations
because of interstial chiasmata. Long chromosomes with median centromeres
tended to form ring-rod and both types of tandem coni’igurations. The
ring-rod and tandem types were most frequently observed in Ganna (7)*

Datura (7* 9), Hordeum (63, 64), iolium (36), Primula (17), and 3ecale (29),
while triradial and triple-arc associations were rare or not found.

The behavior of trivalents and bivalents at metaphase I
has been found to be similar in most triploids and in other plants,
such as autotetraploids, interspecific hybrids and aneuploids, in which
they occur (l, 13, 18, 22, 24, 32, 33, 38, 41, 42, 43, 45, 47, 53, 65).

Normally, at late diakenesis and early metaphase, when the nuclear membrane
disappears, these associations move to the center of the cell and become
oriented into an equatorial plate within the spindle mechanism. At late
metaphase and early anaphase the trivalents usually disjoin two members
to one pole and the third to the opposite, while the bivalent daughter-
members separate one to each pole. According to some investigations,
members of a trivalent may lag and divide equationally at anaphase (20, 36, 64 ).

On the other hand, considerable variability has been found in
the behavior of univalents. Since there are similarities of univalent
behavior in triploids, interspecific and intergeneric hybrids, and

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aneuploids, pertinent information from these sources will be discussed.

The following general description of univalent behavior has
been given by Darlington (20): "Unpaired chromosomes usually lie at
random on the spindle at metaphase. They do not move towards the equator
as early as the paired chromosomes. It is sometimes stated that unpaired
chromosomes lying to one side of the plate are moving to the pole
in advance of the bivalents at anaphase, but this conclusion is unjustifiable.

Their position is due to their never having reached the plate, and they
actually do not move until after the bivalents have divided. When the
paired chromosomes begin to separate at anaphase unpaired chromosomes
follow one of two courses: (l) those lying far away from the equator are
included with the group of daughter bivalents passing to the nearest pole;

(2) those lying near the equator move on to the plate, orientate themselves
axially, and divide after a short interval into their two chromatids,
which then pass to opposite poles as in mitosis," Based on Kihara's
study of Triticum - Aegilops hybrids (Kihara, H. Genomanalyse bei Triticum
und Aegilops . I and II. Cytologia, 2. 1931*)> Darlington concluded that
univalent behavior was chiefly of the second type and that "variations
commonly observed in univalent behavior are probably due to various degrees
of delay in the movement of univalents relative to those of the bivalents."

The behavior of univalents in wheat monosomies as described by
Smith et al. (57) and Sears (55) was similar to Darlington's second type.
However, numerous descriptions of their behavior in various triploids and
interspecific and intergeneric hybrids have indicated that both types may
occur in the same stock.

In a series of papers on interspecific Triticum hybrids Melburn

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and Thompson (34) 9 and 'Thompson (59* 60) reported that univalent behavior
in these hybrids followed a generally consistent pattern. At metaphase
the univalents were more or less scattered throughout the cell, a few
being observed near or on the plate together with the bivalents. After
division of the bivalents the majority of univalents moved to the equator
and divided equationally, the two halves separating to opposite poles.

The remaining univalents did not move to the plate but joined the bivalent-
halves at the nearest polar group. Thus, late anaphase polar groups
consisted of bivalent-and univalent-halves and undivided univalents.

Similar behavior of univalents was observed by Nishiyama (43) in an
interspecific triploid hybrid of Avena and by Sax (53) in a pentaploid
emmer - vulgare wheat cross. The latter paper also included a study of the
hybrid Triticum monococcum X turgidum in which it was found that the
univalents usually lay at or near either pole. In a few cases they moved
to the plate and divided after the bivalents. This description is in
contrast to that which has been given by Thompson (59) for the same hybrid,
as already discussed, but involving different varieties. Sax and Sax (54)
observed that in the intergeneric cross Aegilops cylindrica X Triticum
vulgare the univalents remained at or passed to the poles without dividing
at first division.

Myers (40) and Myers et al. (41) concluded from observations
of meiosis in diploids and autotetraploids of Lolium perenne , and several
other grasses, that some univalents were oriented on the plate with the
bivalents and multivalents before anaphase and that others were scattered
throughout the cell during metaphase but became oriented some time before
completion of anaphase, after which they divided equationally. The few

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unoriented univalents that -were left intact in the cytoplasm formed
micronuclei later. The descriptions of univalent behavior in autotet-
raploid Secale cereale that have been given by Chin (14) and O'Mara (45)
agree closely with that for Lolium .

Gajewski (24) developed a series of Geum interspecific hybrids
with increasing numbers of univalents and studied their behavior at
metaphase - anaphase. Considerable irregularity was found in their
behavior. In two hybrids with the fewest number of univalents, two to
seven univalents were rarely found on the plate at metaphase, and most
passed undivided to the poles at anaphase. The remainder were left at
the plate at late anaphase where they either divided or passed whole to
one of the poles. In two hybrids with 14 and 21 univalents, respectively,
the univalents were scattered over the whole spindle. Later they
congressed at the equator to form a more or less regular ring about
the plate. At anaphase, after separation of the bivalents, all of the
univalents rested on the plate. Their division and movement was very
irregular; some divided; others did not; and many were omitted from the
daughter nuclei. In the fifth hybrid, possessing 42 univalents, three
groups of chromosomes tended to be formed, one at each polar end and
one at the plate composed of a few bivalents and univalents. After division
of the bivalents at anaphase the behavior of the univalents depended on
their position on the spindle. Those on it moved without change to the
nearest pole, while those on the equator appeared to stretch but moved as
whole bodies to the poles. Gajewski attributed the differences of
univalent behavior in the different hybrids to l) differences in genotypical
constitution (an important factor in meiotic pairing) and 2) to different
numerical relationships between univalents and bivalents - the more

bivalents on the metaphase plate, the greater the proportion of univalents
that became oriented at metaphase - anaphase,

A discrepancy between the relative proportions of metaphase
univalents and anaphase laggards can be calculated from the data given
by Boyle and Holmgren (ll) who studied the hybrid between Agropyron
trachycaulum (2n = 28) and Hordeum .jubatum (2n = 28). An average of
13. & univalents occurred at metaphase, which was approximately one half
of the entire chromosome complement, while at anaphase only 3*51 laggards
were observed (this value is calculated from data given in Table IA of
their report). In the amphiploid of this hybrid an average of 1.3
univalents were observed at metaphase and 1.4 laggards at anaphase (3).

The data from this hybrid and its amphiploid tend to substantiate
G-ajewski's second conclusion.

Most reports of univalent behavior in triploids, particularly
those from early investigations, are largely descriptive. In triploid
asters all of the univalents divided at first division (4)> while in
Solanum tuberosum they lagged but did not divide (35). The absence of
lagging at second division further indicated that in the latter species
univalents seldom, if ever, divided at first division. Although lagging
univalents occurred occasionally in triploid tomato, they rarely divided
at anaphase (31). In triploid lilium Chandler et al. (13) found that
univalents were usually together with the trivalents and bivalents at the
plate. Most frequently they lagged and divided equationally at anaphase
after the trivalents' and bivalents had separated. McClintock (33) stated
that in triploid corn univalents oriented on the plate probably divided
at the same time as the trivalents and bivalents, while lagging univalents
separated later. More precise and conclusive information on univalent

( . -

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behavior in triploids has been obtained in more recent studies.

Approximately 43 per cent of the lagging univalents of triploid
rye divided at anaphase (29). In triploid Phleum pratense (44) an average
of 4*68 univalents occurred at metaphase, but an average of only 1.81
univalents lagged and divided at anaphase. According to the data given
by Punjasingh (47) on triploid corn, approximately 49 per cent of the
microsporocytes had one or more univalents at metaphase, while only
about 25 per cent had one or more laggards at anaphase. The writer's
calculations of Punjasingh's data indicate that about 55 per cent of all
metaphase univalents lagged and divided equationally at anaphase.

Tsuchiya (64) found an average of 2.33 univalents at metaphase of a
- hybrid triploid barley and an average of 1.46 laggards at anaphase. Of
the latter, about 36 per cent were calculated to be derived from "improper”
disjunction of trivalents and the rest from univalents oriented on the
equatorial plate at metaphase. The results indicate that in these
triploids a relatively large proportion of metaphase univalents did
not divide at anaphase. In contrast to this behavior, Myers (38)
calculated that in triploid Lolium perenne an average of 1.33 laggards
occurred at anaphase as compared with 0.93 univalents at metaphase. The
excess of anaphase laggards was attributed to "improper 11 disjunction of
trivalents. In triploid hyacinths Darlington (18) also attributed an excess
of anaphase laggards when compared with the number of metaphase univalents
to imperfect disjunction of trivalent associations, although no supporting
data were given.

In normal diploids the equational split at anaphase occurs
simultaneously in all daughter-members of disjoined bivalents. They are
then termed diads. Aase (l), Darlington (20), Gajewski (24)* and love (32),

' Xu . . . ,

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;

[

stated that the equational split occurred simultaneously in all unpaired
chromosomes and disjoined members of bivalents* In contrast to this
behavior Myers (38) noted that many univalents oriented on the metaphase
plate showed the 'split* before initiation of anaphase, while those off
the plate did not* Aase (l) concluded from her studies on the cytology
of numerous cereal hybrids that "The behavior of the univalents depends
largely on their location on the spindle at the time of the equational
split. The equational split may, however, overtake them at any location
on the spindle, and consequently, if many univalents are at the equator
at this critical time many univalents will divide.”

It is generally assumed that univalents of aneuploids and
interspecific hybrids and the extra set of chromosomes in triploids,
whether they occur in associations as trivalents or unassociated, are
distributed at random to the poles during meiosis (20). Some authors
have claimed or assumed random distribution of univalents in certain inter¬
specific gramineous hybrids without giving statistical data (l, 27, 43,

53, 54, 60). O'Mara (45) also assumed randomness of univalents in
tetraploid rye. Anaphase distribution of chromosomes in triploid corn
"appeared” random (45)* Although Tsuchiya (64) presented data on the
observed distribution of the chromosomes at anaphase of triploid barley
no statistical comparison with a binomial distribution was given. Visual
inspection of his data indicates that several distribution classes were
not in accordance with randomness, perhaps because of the relatively
small numbers of cells recorded. Chromosome distribution at anaphase has
been described as approaching or resembling randomness by comparing, without
statistical tests of significance, observed frequencies with frequencies

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calculated according to the binomial in triploids of Ganna ( 5 ), Datura ( 8 ),
Lycopersicuni (31 ) } and Zea (47)*

To date the hypothesis of random distribution of the third set
of chromosomes in triploids has been adequately tested in only two
species. Satina and Blakeslee (51) recorded the distribution of
chromosomes at first division in 1000 microsporocytes of triploid Datura
stramonium (3x = 36). They found an excess of the more extreme anaphase
groupings, 12 - 24 , 13 - 23 , 14-22, 15 - 21 , and a deficiency of the intermediate
groupings, 17-19 and 18 - 18 . The discrepancies were statistically
significant. The authors concluded that "Despite the lack of direct
evidence from other forms than Datura , it seems probable that the
divergence of the assortments at the I division in P.M.C. from calculated
values is of general occurrence and is to be attributed to the nature
of chromosomes and the mechanisms involved in their movements at
division." Myers (39) tested the randomness of chromosome distribution
at anaphase of triploid Loliurn perenne (3x = 21). The statistical
■treatment of the data obtained from 2,494 metaphase and 1636 anaphase
microsporocytes indicated that 1 ) at metaphase "unoriented univalents lie
in the microsporocyte at random relative to one another and to the
equatorial plate", and 2 ) "The distributions at anaphase I also were
consistent with the hypothesis of chance position of the unoriented
metaphase I univalents and random assortment of the extra chromosomes of
the trivalents." Thus, the behavior found in triploid Loliurn differed
from that in triploid Datura.

Descriptions of univalent behavior during the stage from late
anaphase to early interphase were similar in several papers reviewed. In

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Triticum (53, 60) and Avena (43) interspecific hybrids, autotetraploids
of Secale (45) and Loliurn (4l)* as well as triploid Lilium (13), the
univalents that were oriented at the plate divided after the bivalents
and other associations. The resulting univalent-halves usually moved
to opposite poles in time to be included in the polar groups at telophase;
if not, they were excluded to form micronuclei in the cytoplasm.

Aase (l) and Kelburn and Thompson (34) occasionally observed that after
division of a univalent lying off the plate both daughter-halves moved
to the same pole. Chromosome fragmentation has been attributed to
misdivision of lagging univalents at telophase (32, 55) and to lagging
univalent-halves being cut into two by cell wall formation at early
interphase (18, 42, 63). Univalents beyond the influence of the spindle
have been observed to form microcytes on the periphery of the cell
(7* 13* 14* 16, 23, 33* 34* 64 ). 'The univalent within such microcytes has
been found to carry on division and pass through stages comparable with
the two main daughter cells (16).

Restitution nuclei formed at the conclusion of first division
have been observed in triploids of Datura (8, 9) and Tulipa (42).

Second Division

Myers (40) and Smith et al. (57) observed that at second division
all diads usually aligned to form an equatorial plate in each daughter
cell and then divided equationally. The univalent-halves, derived from
the previous division of univalents, lagged at the equatorial region
during anaphase - telophase and either moved to the poles or were excluded
to form micronuclei. In Avena hybrids (43) and in several other grasses
that possessed univalents (41) some of the diads and univalent-halves
remained at the poles to be included in the nuclei at telophase. Lagging

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univalent-halves have been observed to misdivide at telophase (55) •

Without providing data Melburn and Thompson (34), Nishiyama (43),

0 1 Mara (45), Sax and Sax (54), and Thompson (60) assumed that univalent-
halves at anaphase - telophase passed at random to either pole. Muntzing (35)
and Thompson (6l) noted that restitution nuclei occurred following
second division.

As a result of lagging chromosomes and their fragmentation
at both divisions of meiosis a large proportion of the microspores of
triploids have been observed to contain one or more micronuclei
(13, 29, 38, 63, 64)* The proportion of ’good’ pollen has been found to
vary among different triploids. From eight to nine per cent of triploid
Lilium pollen germinated on artificial media (13), while from five to
15 per cent of triploid Datura pollen germinated on 3x stigmas (12).
Approximately 92 per cent of the pollen of triploid corn (47) and
54 per cent of the pollen of triploid barley ( 64 ) was found to be well
filled with starch. Similarly, the fertility has been noted to vary.

Triploids of Lycopersicum esculentum (31) and Secale cereale (29) were
completely self-sterile. Selfed triploid Lilium . (13) had 20 per cent of
the fertility of the diploid. Tsuchiya ( 64 ) found a seed set of 19 per cent
on open-pollinated triploid barley ; the fertility was increased by hand-
self ing and by crossing with pollen from diploids. Punjasingh (47)
determined that 11 per cent of the florets of triploid corn set seed,
presumably when open-pollinated.

Functional Gametes of Triploids

In all reports reviewed (8, 12, 21, 22, 26, 30, 33, 38, 47, 51,

' »•

4 ' ■ 'j-

b X

52 , 63) a marked discrepancy was noted in the frequency of the chromosome
numbers in functional pollen and eggs of triploids when compared with a
binomial distribution. These included triploids in which distribution
of the chromosomes to the poles at meiosis had been found to be random.

The number of extra chromosomes in functioning gametes has been found to
vary among species. It has also been noted that extra chromosomes were
transmitted with a higher frequency through female than male gametes
(12, 22, 33)* In triploids of Zea (33* 47) and Petunia (21) pollen with
the haploid number or with one extra chromosome functioned exclusively,
except for the occasional one with the diploid or near diploid complement.
Pollen of triploid Secale (29) and triploid Lycopersicum (31) was
nonfunctional on both 3x and 2x stigmas. Functional eggs of triploids
of Datura (8, 12), Lolium (38), and Lycopersicum (30) were found to possess
up to two or three extra chromosomes. In addition to these extra
chromosome types, a relatively low proportion of the functional female
gametes of triploid Nicotiana (22) and triploid Zea (33* 47) had
•intermediate numbers ranging between the haploid and diploid complement.
Some triploids, therefore, tended to have progeny with chromosome numbers
approaching almost exclusively the diploid number, while others produced
additional types having intermediate numbers of the entire possible range
but with a much lower frequency than theoretically expected.

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MATERIALS AND METHODS

The triploids described in this study were obtained from two
sources. Three triploid plants were found in a large F 2 field population
derived from intervarietal F]_ hybrids, which as germinating seeds had
been treated with colchicine. Another group of 13 were found among
4500 progeny head-rows of a highly homozygous line of the variety
Gateway which had been treated with various antibiotics, fungicides and
insecticides in the previous generation. The original objective of this
material was to test the mutagenic properties of these compounds. All
but three of the triploids were discovered during the flowering period
because of their high sterility, exhibited at this stage by the open
florets. The three triploids of hybrid origin, and 10 of the Gateway
triploids were eytologically identified by chromosome counts of pollen
mother cells or of mitotic divisions in ovary tissue. 'The remaining
three, detected at harvest time because of their very low seed set, were
assumed to be triploid since they produced aneuploid offspring similar to
those obtained from the eytologically proven triploids.

Secondary tillers were collected from the Gateway triploids for
cytological study of microsporocytes. They were fixed in Carney 1 s 6:3:1
at room temperature for two to three days and then stored under refrig¬
eration in the same solution for periods up to 15 months before being
examined.

Temporary aceto-earmine preparations were made for studying
chromosome behavior. Mature pollen grains were stained with an iodine
solution to determine the proportion of ’good' pollen.

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Cytological data were recorded for three or four of the Gateway
triploid plants. Although data for each were recorded separately, no
attempt was made to analyze them individually because of insufficient
material available from any one plant. Only those metaphase I and
anaphase I cells in which all chromosomes and their associations could
be clearly distinguished were recorded. Similarly, only microspore
tetrads with the surrounding wall intact were recorded.

Seeds harvested from the triploid plants were sown either in
pots in the greenhouse or in field plots. Chromosome numbers of the
resulting progeny were determined by counts in pollen mother cells or in
somatic ovary tissue. The latter method was used because some of the
plants tillered poorly and were highly sterile. To obtain a maximum
number of seeds from these, a few florets only were removed for cytological
examination from a head as it emerged from the leaf sheath.

Microscopic observations and photomicrographs were made using a
Ziess Cpton microscope fitted with apochromatic lenses and a reflex plate
camera attachment.

OBSERVATIONS AND RESULTS

Occurrence of Triploids

Three triploids were found among approximately 3000 progeny
head-rows of an F 2 hybrid nursery derived from F^ plants treated with
colchicine. One of the triploids was found among the progeny of a plant
which also produced diploids and tetraploids, while the other two occurred
in the progeny of different plants in which no tetraploids were observed.

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Three of the 13 triploids found in the Gateway material occurred
among check progeny rows. This would indicate that some of these triploids
originated spontaneously* without treatment effect. Except in one case
where two triploids came from the same mother plant* the sibs of all
triploids were fully fertile* and therefore* presumably diploid. Wo
attempt was made to obtain a reliable estimate of the frequency of
occurrence of triploids among treated rows. However* an estimate was
calculated for check rows. Four hundred and thirty-three check rows*
which constituted approximately one tenth of the entire population*
were closely observed plant by plant for sterility* as an indication
of possible triploidy* several times during the flowering period and
again at harvest time when low set of seed could be detected. Under this
careful scrutiny three cytologically identified triploids were found at
flowering time. 'The average number of plants per check row was estimated
from an exact count of 82 rows. The total number of plants in the 433
check rows was then estimated to be approximately 18*500. From this value
‘the frequency of spontaneous triploids was determined to be about one
in 6000 plants.

Experimental Production of Triploids

When it was observed that triploids occurred spontaneously in
check rows, an attempt was made to produce them experimentally by hand-
pollinating Gateway autotetraploids with diploids of this variety. One
poorly developed* inviable seed was obtained from 242 pollinated florets.

Morphology of Triploids

The triploids were indistinguishable morphologically from diploid

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sibs. As stated previously, they were noted only because of the high
degree of sterility characteristically evident at flowering time when
the florets remained open for several days beyond that normally observed
in diploids. Fig. 2 shows representative plants of diploid, triploid,
and tetraploid Gateway.

Meiosis in Triploids

Observations were made on chromosome behavior at meiosis in
microsporocytes at metaphase I and subsequent stages of the Gateway
triploids. Data from several plants were combined for analysis.

Iletaphase I

At metaphase I the pollen mother cells contained various
combinations of trivalents, bivalents, and univalents, as shown in Table I.
The most common combinations were 6 -q-j + ljj + lj and + 2 tj + 2 j, each

occurring with a frequency of about 30 per cent. No cells with
P~~ T + + 7j were noted.

All types of trivalents that are possible from normal pairing
of three homologous chromosomes were observed - tandem-V (Figs. 4, 5, 7),
tandem-chain (Fig. 7), ring-rod (Figs. 3, 4, 5, 6, 7, 8), triple-arc
(Figs. 3, k) } and triradial (Fig. 5). Table II shows that slightly
more than 50 per cent of the trivalents were the ring-rod type. The
triradial type was least frequent, only 14 being observed in 865 cells.

The bivalents were either closed or open, with 5*71 per cent being
of the latter type. An average of 5.22 trivalents and 1.78 bivalents
per cell occurred.

In addition to the above expected associations of homologous
chromosomes and their various combinations, the following unusual sorts

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TABLE I

Frequencies of combinations of various chromosome
associations at metaphase I

_ Frequency _ ( _

Combination of % of

associations _ No. of cells _ total cells

7 III

159

14.96

6 III

+ lj-j-

+ 1 i

328

30.85

5 III

+ 2 n

+ 2

317

28.82

^III

+ 3 n

+ 3 i

171

16.09

3 III

+ k xi

+ h

6.49

2 m

+ 5 n

+ 5

1.41

1 iii

+ 6 ii

+ 6 i

0.38

7 ii

+ 7 i

0.00

1063

100.00

were observed among 1091 cells examined:

1. A quadrivalent in each of three cells (Fig. 8).

2. Three hexaploid cells (2n - 42) having only trivalents,
bivalents, and univalents.

3. One cell with an extra bivalent (2n = 23).

4. Seven trivalents and a fragment in a cell.

5. One microsporocyte deficient for three chromosomes (2n = 18).

6. One cell with five trivalents, one bivalent, and four
univalents (Fig. 7).

7. Seven cells with a 1 side-by-side 1 , and seven cells with an
'end-to-end 1 association of two univalents. These have been also termed
pseudobivalents and secondary associations (46). These associations were
observed to lie off the plate in all but one cell.

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TABLE II

Frequencies of various types of trivalents and bivalents
observed in 865 metaphase I cells

Frequency

Configuration

Total no.

% of total

Average per cell

Trivalents

Tandem-V

1337

29.63

1.54

Tandem-chain

632

14.01

0.73

Ring-rod

2342

51.89

2.71

Triradial

0.30

0.02

Triple-arc

188

4.17

0.22

4513

100.00

5.22

Bivalents

Closed

1454

94.29

1.68

Open

5.71

.10

1542

100.00

1.78

Behavior of Trivalents and

Bivalents.-

At metaphase I the

and bivalents usually were observed to form an equatorial plate.
Occasionally one or two of these associations were seen to lie off the
plate. Bivalent daughter-halves were oriented with the spindle fibre
attachment regions toward opposite poles, while orientation of trivalent
members depended on the type of configuration. Tandem-V, triple-arc,
and triradial types were oriented two and one to opposite poles
(Figs. 5 } 6, 7). Tandem-chain configurations were found to lie with
a member oriented to each pole and the third member interposed between,
unoriented (Fig. 7)* 'The ring-rod type was most frequently found to be
oriented with two members toward one pole and the third to the other

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Fig. 2. Diploid, triploid and tetraploid Gateway*
Fig. 3* Diakenesis with + 4tj 4* 4-- and
showing triarc configuration (arrovj)*

Fig. 4* Metaphase I cell with 7 ixi> one tandem-V,
four ring-rod and two triple-arc (arrows)
c onfigurations.

Fig. 5.

Fig. 60
Fig. 7.

Fig. 8 .

Metaphase I with 3 t ^j + An + A ; two
univalents on plate, one being ^
oriented; univalents off plate dis¬
tributed 0 - 2 .

Metaphase 1 showing two triradial
trivalents (arrows) and 1-1
distribution of univalents.

Metaphase I with 5jH + ijl + one
tandem-V, and two ring-rod trivalents;
one unoriented univalent on plate and
three distributed 1-2 off plate.
Metaphase I showing a quadrivalent
(arrow)•

■

(Figs, 4, 5> 6, 7)* Occasionally, however, the rod meniber of this type
lay more or less parallel to the plate without orientation of the
attachment region to either pole.

Behavior of Univalents .- The position of the univalents in the cell
varied considerably. They were observed to lie at the polar regions, in
the vicinity of, or on the equatorial plate (Figs. 5, 6, 7). Those at
the poles appeared to be oriented haphazardly, while those at the plate
often were noted to lie parallel to it. Univalents not on the plate or
at the polar regions were observed in various positions, extending from
near the main group of equatorial chromosomes to the poles, randomly
oriented.

In order to determine the significance of the position of
univalents in the cell at metaphase in relation to their subsequent
behavior, the following information was obtained on univalents: 1) their
position, on or off the plate: 2) orientation of those on the plate ; and
3 ) the distribution to opposite sides of the plate of those not located
on it. A univalent was regarded as being on the plate when observed
to lie in the equatorial region. This region was considered to extend
from one side of the cell to the other along the equatorial axis and
approximately two thirds the length of a tandemr-chain trivalent along
the polar axis. As an example, in Fig. 5 two univalents are on and two
are off the plate. The position of some univalents was not clearly defined.
These were more or less arbitrarily recorded into either group on the
assumption that univalents so observed would be entered with equal
frequency into both groups. For example, in Fig. 7 three univalents
were recorded as off and one as on the plate. Univalents on the plate
were further classed as oriented if lying more or less parallel to the

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equatorial axis and as unoriented if in any other plane (Figs. 5* 7)*

The proportion of univalents on the metaphase plate for each
of the seven combinations of chromosome associations is given in Table III.
The per cent of univalents on the plate in each class varied from 42.38
to 23*00. The latter value cannot be regarded as reliable, since only
four cells were recorded for this class of ljjj + 6^ + 6^.. The
proportion of univalents on the plate in the remaining six classes
ranged from 42.38 to 32.23 per cent. From the data in the table the
average number of univalents per cell was calculated to be 1.74* and
the average number of these on the plate was 0.66, or 37*73 per cent.
Expressed in another way every 100 cells contained 174 univalents of which
66 were on the plate.

TABLE III

Proportion of univalents on the plate at
metaphase I

Combination of
chromosome
associations

Total no.
of cells
examined

Total no.
of

univalents

Univalents on
plate

ToF

No. total

7 m + °n + °i

159

—

6 iii + hi + 1 i

328

139

42.38

5 iii + 2 ii + 2 i

317

634

234

36.91

4in + 3 JX + 3 J

171

513

196

38.21

3 III + 4 II + 4 i

276

32.25

2 III + 5 II + 5 I

45*33

hn + 6 II + 6 I

25 .OO

Total

1063

1850

698

Av.37.73

For each combination of chromosome associations, univalents
on the plate -were further classified as oriented or unoriented, as
indicated in Table IV, The per cent of total univalents oriented on the
plate ranged from 25.10 to 16.67* with an average of 20.52. It was
calculated that an average of 0.37 oriented univalents per cell occurred
or, stated in another way, 37 univalents in every 100 cells. Approximately
54 per cent of all univalents on the plate were oriented. A summary of
the data on the frequencies of the various classes of univalents is
given in Table V.

TABLE IV

Proportion of univalents oriented on the
plate at metaphase I

Combination of
chromosome
associations

No. of
cells

Total
no. of
uni¬
valents

Univalents
on plate

Total No.

no. oriented

Oriented

of total

univalents

fo of

total
on plate

7 +0

'iii ii

+ 0.

117

—

6 m + x n

+ 1 i

243

100

25.10

61.00

5 m + 2 n

+ 2 i

246

492

187

100

20.33

53.48

4 TTT + 3
III Ii

+ 3 x

135

405

155

20.49

53.55

3 +4

III II

+ 4

228

16.67

47.50

2 m + 5 u

4 5

20.00

52.00

1 m + 6 n

+ 6 I

—4

-2k

-k

16.67

66.67

Total

815

1457

553

299 Av,

.20.52

54.07

■

> • ' •••

TABLE V

Summary of univalent classes at metaphase I

Glass

Frequency

Per 100
cells

% of total

univalents

From Table III

Univalents on plate

37.73

From Table IV

Oriented univalents
on plate

20.52

The proportions of cells having various numbers of univalents
on the metaphase plate -was also recorded (Table VI), since, as -will be
shown later, these may influence the proportion of anaphase cells with
various numbers of lagging univalents.

TABLE VI

Frequency of metaphase I cells with
various numbers of univalents on the plate

No. of univalents No. of $ of total

-qel. slate.CLalls cells,

557

52.40

354

33.30

119

11.20

2.54

0.47

0.09

0.00

_ 0

0.00

1063

100.00

To test the assumption that univalents off the equatorial

plate occurred on opposite sides of the plate at random, the observed

distribution frequencies for each class of univalents off the plate can

be compared with the calculated* For example, where two univalents

occurred off the plate, they would be expected to lie on the same side

(0-2 distribution) and one on each side (l-l distribution) in equal

frequencies. Similarly, with three univalents off the plate the 1-2

and 0-3 distributions would be expected in a 3il ratio. In cells with

four univalents off the plate distributions of 0-4, 1-3 and 2-2 should

be expected in a ratio of 1:3*4. The X analysis for observed and
calculated distribution frequencies for all classes of univalents off the
plate is given in Table VII. In the table the data are arranged in
classes according to the total number of univalents per cell, each of
which is subdivided according to the number of this total that were off
the plate. In all classes except one the fit of observed to calculated
frequencies is satisfactory. The exception is for the class in which
two univalents of a total of three occurred off the plate. A probability
level of less than 0.01 indicates that the distribution of the 0-2 and
1-1 classes deviated significantly from the expected 1:1 ratio.

The data from all identical distribution classes in Table VII
were combined so as to obtain a single test of fit of observed to cal¬
culated frequencies of the various distributions. For two univalents
off the plate the fit to a 1:1 ratio of 0-2 and 1-1 distributions was
poor (P = 0.02 - 0.01). This is due to the discrepancy, noted above
in Table VII, for the class in which two of a total of three univalents
occurred off the plate. The combined data for the distributions of
three univalents off the plate (0—3 and 1-2) gave a good fit to a 1:3

r . a-;?'!

■ • -

• 1 ■ • ■ 1 ' ; ; •

’

- ; '

. , . . ■ . • > . . . ■

.....

. . j. a ■ ■ ■■ ; :

. '

; . : ?; .r ...

.. .

■

;v«.i

• ^ c

! ... '/W ' '

■

TABLE VII

X analysis of observed and calculated frequencies
of distributions of metaphase I univalents to opposite sides

of the plate

Total no. of
univalents
per cell

Univalents

plate

off

No. of cells

Total

no.

Distri¬

bution

Observed

( 0 )

Calculated ( 0 -C)

67.00

1.21

67.00

1.21

134

134.00

x 2 - 2.42

0.20-0.10

12.00

0.33

36.OO

1.11

4B.00

X 2 = 1.44

0.30-0.20

33.00

3.67

33.00

3.67

66.00

X 2 = 7.34

<0.01

2 . 3 s

0.16

9.50

0.03

7.12

0.18

19.00

X?= 0.37

0.70-0.50

—

5.25

0.58

Hill

2M2

21.00

X 2 = 0.77

0.50-0.30

10.00

0.40

10.00

20.00

X 2 = 0.80

0.50-0.30

ratio (r =0.95 - 0.50).

The data in Table VII were combined in a second manner to test
the randomness of the distribution of univalents to opposite sides of the
plate. Similar groupings of univalents, ie., 0, 1, 2, 3 } k> from each

of the distributions, ie., 0-2, 1-1, 0-3, 1-2, etc., -were combined for
all observed and calculated values, respectively, and then subjected to

a X analysis. A probability level of 0.10 - 0.05 for the combined
data in Table VIII further substantiates the assumption of random
distribution of univalents to opposite sides of the plate.

TABLE VIII

X analysis of total frequencies of univalent
groupings located off the plate at metaphase I

Frequency

Univalent
grouping .

Observed

frequency

... -(o)

Calculated

frequency

. . CO ..

{o=cf_

148

129.63

2.60

246

281.25

4*42

192

175.99

1.46

26.75

.02

616

P = 0.10

2.38

6X6.00

- 0.05

.16

X = 8.66

The overall results of the statistical analysis of the data
on univalents occurring off the equatorial plate indicate that in
general they -were distributed to opposite sides of the plate at random
during metaphase I.

Anaphase I

At anaphase I all polar groups contained not less than seven
diads nor more than 14. Presumably^ therefore, one chromosome from each
bivalent and trivalent invariably moved to each pole. From zero to four
univalents were observed to lag at the equatorial region after separation

■

and movement to the poles of the daughter chromosomes of the bivalents
and trivalents. At this time all diads appeared equationally ’split’,
■whether found at the poles or lagging at the plate (Figs. 9> 12).

Lagging univalents usually divided equationally at late anaphase, and the
daughter-halves then moved to the poles (Fig. 9* 13)* In a small proportion
of cells lagging univalents were observed to misdivide (Figs. 10, 11).

Also, in a few cells one and occasionally two univalents were noted to
lie on the extreme periphery of the plate or polar group (Fig. 12),
apparently beyond the influence of the spindle mechanism. These did
not divide nor were they included in the main polar groups at anaphase
or telophase (Figs. 12, 14).

It was calculated above that for every 100 cells at metaphase I
there were a total of 174 univalents. Of these 66 were on the plate,

37 being oriented (Table V). From the data in Table IX it was further
calculated that an average of 80 lagging univalents were present in
every 100 cells at anaphase I, which is 45*97 per cent of the total
number of univalents at metaphase I. This indicates that less than one half
of the metaphase I univalents lagged and divided at anaphase I. Although
this value is greater than the proportion of univalents on the plate at
metaphase, that is 37*73 per cent (Table V), it does approach the latter
value rather than 20.52 per cent (Table V), the proportion of oriented
univalents on the metaphase plate. The evidence supports the assumption
that all univalents on the plate at metaphase I, including those recorded
as unoriented, lagged and divided at anaphase I. Presumably, univalents
classed as unoriented became oriented by the time anaphase was initiated.

The higher percentage of univalents found to lag at anaphase could be
explained as the result of the recording system used. Conceivably, some

■

•.

.......

.... . . ..... . . .

■■..< i,;.. .;. iV'i;-; •• ..i.

of the univalents lying close to the plate, and recorded as off, later
moved on to it and lagged at anaphase, after division of the bivalents and
trivalents. .

TABLE IX

Frequency of anaphase I cells containing
various numbers of lagging univalents

No. of
lagging
univalents
ner cell

No. of
cells

% of
cells

Total
no. of
laggards

191

48.23

131

33.08

131

12.88

102

2.53

2.78

0.50

0.00

396

100.00

317

Visual comparison of Tables VI and IX indicates that the
proportions of cells with various numbers of univalents on the plate
at metaphase I approach the proportions of cells at anaphase I with
corresponding numbers of lagging univalents. On the basis of the
assumption stated previously that only those univalents on the metaphase
plate lagged and divided at anaphase and the evidence already presented,
the observed frequencies of anaphase I cells with zero to five laggards
(Table IX) were compared with frequencies calculated from the proportion of
metaphase I cells having the corresponding numbers of univalents on the

*-rj hr| hrj t-xj *ij

1. ■ • - — -

fy* *

10 £

■

H •*

- * *

Fig. 9* Anaphase I showing 8 - 11 distribution, one lagging diad,
and two lagging daughter-univalents.
ig.10. anaphase I with lagging univalent irdsdividing transversely,
ig.ll. Anaphase I with two lagging univalents ndsdividing.
ig.12. Anaphase I cell with two peripheral lagging diads.
ig.13. Early telophase 1 with lagging daughter-univalents.
ig.14* Telophase I cell showing appearance and location of
peripheral diad.

plate (Table VI). The X analysis of the data in Table X indicates a

discrepancy between the observed and calculated values* The poor fit

(P =< 0.001) is due almost entirely to the excess of cells observed in

the classes with four and five laggards, as indicated by a probability

level of 0*70 - 0*50 when the remaining classes were subjected to a
2

separate X test. The discrepancy may be due to the relatively small
number of cells observed in the two classes and also, as previously
stated, to error in the system used for recording the position of
univalents as either off or on the plate at metaphase I, which could
have contributed to an inaccurate estimate of calculated values.

TABLE X

X analysis of observed and calculated frequencies
of lagging univalents at anaphase I

No. of lagging
univalents

Observed

(0)

Calculated

(c)

O I

191

207.50

1.31

131

131.37

0.01

44.35

1.00

10.06

0.00

1.86

44.91

0.36

7.47

396

396.00 X 2 =

54.80

P 0,

.001

The results nevertheless indicate that in the majority of cells
univalents located on the plate at metaphase I lagged at anaphase I,
after division of the bivalents and trivalents.

Cn the evidence already presented to indicate that the dis¬
tribution of univalents on opposite sides of the metaphase I plate
tended to be. random (Tables VII, VIII) and on the assumption that the
third member of a trivalent passed to either pole also at random, it
appears valid to compare the observed frequencies of chromosome dis¬
tributions to the poles at anaphase I -with that expected for none and
various numbers of lagging univalents, according to a binomial frequency.
In cells with no laggards at anaphase the distribution of the extra
seven chromosomes can be calculated from the expansion (g + g) .

For cells with one laggard the distribution of the remaining six should
be according to the expansion (g •+ g)^ 1 , for cells with two laggards
(2 + 2)^ , f° r those with three laggards (g + for cells with four

lagging univalents (g + g)^, and for those with five laggards (g + g)^.

No cells with more than five lagging univalents were noted in 369 that

were examined, ‘The observed and calculated frequencies of anaphase

distributions and analysis by the X method is presented in Table XI*

No analysis is given for cells with five laggards, since there were only
two in each category, one for each of the two possible types of dis¬
tributions, 7-9 and 8-8, which are expected in a 1:1 ratio. The
results of the analysis indicate that the chromosomes were distributed
to the poles in a binomial frequency, except for the category with two
laggards. 'The discrepancy here is due to the 7-12 and 8-11 dis¬
tributions. When these were combined and tested with the remaining class
a good fit of observed to calculated was obtained (P = 0.30 - 0.20).

To test further the randomness of chromosome distribution at
anaphase I, the observed data on the one hand and the calculated on the
other from all distributions in Table XI were combined to form eight

■

TABLE XI

X analysis of observed and calculated frequencies
of chromosome distribution classes at anaphase I

No, of
laggards

per cell_Distribution classes and frequency of cells

7-14

8-13

9-12

10-31

Total

Observed (0)

100

191

Calculated (C)

(0 - c r

2.99

20.89

62.67

104.45

191

0.34

0.21

0.03

0.19

0.77

P =

0.90 -

0.80

7 - 13.

8-12

. .9. - 11

10 - 10

Total

Observed (0)

131

Calculated (C)

(0 - c) 2

4.09

1.07

24.56

0.01

61.41

0.32

40.94

1.22

131

2.62

P -

0.50 -

0.30

7-12

8-11

9-10

Total

Observed (0)

Calculated (C)

3.19

15.95

31.87

(0 - C) 2

2.47

3.02

0.54

6.03

P a

0.05 -

0.02

7 - 11

8-10

9-9

Total

Observed (0)

Calculated (C)

1.25

5.00

3.75

(0 - c) 2

0.50

1.80

2.02

4.32

P -

0.20 -

0.10

7-10

8-9

Total

Observed (0)

Calculated (C)

(0 - c) 2

2.75

0.56

8.25

0.19

0.75

P =

0.50 -

0.30

possible polar chromosome groups. The number of chromosomes per group

ranged in consecutive order from seven to 14. A probability level of

0.80 - 0.70 obtained from the X analysis of the combined data in Table XII
indicates that the proportions of the various groupings occurred in
frequencies expected from random assortment of chromosomes to the poles.

TABLE XII

Total frequencies of anaphase I polar chromosome
groupings

No • of _ Frequency

chromosomes
in group

Observed

(0)

Calculated
. (C)

i2=sr

15.27

0.49

76.64

0.17

167

172.70

0.13

244

225.95

1.44

167

183.05

1.41

90.42

0.14

24.98

0.00

2.99

0.34

792

792.00 X 2 =

4.12

P = 0.80

- 0.70

The overall results of the analysis of chromosome behavior
at anaphase I indicate that the extra set of seven chromosomes ■were
distributed at random to the poles, whether they occurred at metaphase I
as members of trivalent complexes or as univalents lying off the plate.

As mentioned previously in the general description of anaphase,
a small proportion of cells contained univalents that behaved abnormally.

In 396 cells examined, 13 were observed to have a diad lying on the
periphery of the plate or pole. Apparently, these neither divided nor
were included with the main group of polar chromosomes. Another four
cells each had two diads similarly positioned. Altogether, 17 cells or
4.29 per cent had one or two peripheral diads. Misdivision of lagging
univalents was noted in two of 396 cells. In one (Fig. 10) a univalent
was observed to misdivide transversely at the centromere to produce two
equal-armed daughter-univalents. In the second cell two lagging univalents
appeared to misdivide in a manner that would produce single-armed fragments
or telocentrics. In another two cells the daughter-halves of an equationally
split univalent were seen to move to the same pole.

Telophase I

At telophase I the microsporocytes contained two polar groups
of chromosomes with from zero to eight lagging daughter-univalents located
at various positions between (Fig. 13). In some cells one or two peripheral
diads were again observed (Fig. 14). Also, in a few cells misdivision
could be inferred from the centric and acentric fragments that were
observed.

The proportions of cells with various numbers of lagging
daughter-univalents are given in Table XIII. The class with one lagging
daughter-univalent was combined with that for two, since presumably one
of two univalent-halves had already joined a polar group at the time of
observation. The 'undetermined 1 class includes cells with irregular
numbers of lagging univalent-halves and fragments that could not be
definitely placed in the other classes. A legitimate comparison of
observed frequencies of cells having various numbers of lagging daughter-

• .

■

univalents -with expected frequencies calculated from anaphase I data
cannot be made because of the relatively large number of undetermined
cells. However, the proportions of cells with and without laggards
can be validly compared. At anaphase I 4^.23 per cent (Table IX) and
at telophase I 46.92 per cent of the cells showed no lagging. These
values are in close agreement.

table; xiii

Frequencies of cells with various numbers of
lagging daughter-univalents at telophase I

No.

of lagging daughter-univalents

. L .

Unde-

6 8 termined

. Total

No. of cells

198

119

7 1 66

422

% of cells

46.92

28.20

7.34

1.66 0.24 15.64

100.00

Of the 422 cells examined 34* or 8.06 per cent, had one or two
diads located at the periphery. Presumably these had remained in this
position from anaphase through to the end of telophase. Misdivision of
a univalent was observed or was inferred from fragments in eight cells,
or in 1.90 per cent of the total. This value probably is somewhat less
than the actual one because misdivision probably occurred in some of
the cells classed as ‘undetermined 1 .

Interphase

At interphase two large nuclei were usually observed, one in
each of the two daughter cells that resulted from first division. One
and rarely two microcytes were associated with 51* or 9«17 per cent,
of 556 pairs of interphase daughter cells that were recorded (Fig. 15).

Each of two microcytes, associated -with different interphase cells,
had two distinct minute nuclei. The remainder had one. Presumably,
the microcytes were formed by diads previously observed on the
periphery of anaphase and telophase cells. Approximately 57 per cent
of the interphase microsporocytes contained no micronuclei, while
one to six were observed in the remainder. An average of 0.67
micronuclei occurred in each pair of daughter cells.

Second Division

The precise behavior of the chromosomes at second division
could not be clearly observed in the material available. However, a
general description can be outlined from the data obtained.

At metaphase II diad chromosomes were aligned on the plate,
often simultaneously in both daughter cells (Fig. 16). Univalents
derived from equational division of lagging diads at anaphase I -
telophase I were observed lying throughout the cell, off as well as on
the plate. The few fragments that occurred usually were scattered in the
cytoplasm. No lagging univalents or fragments were observed in 45*59
per cent of the pairs of daughter cells examined. A single microcyte
was associated with 8.05 per cent of the pairs of cells. Four of these
21 microcytes had two diads and the remainder had one. All of the above
metaphase II observations are based on data recorded from 261 pairs
of daughter cells.

At anaphase II, diads that were aligned on the equatorial
plate separated and moved to the poles as in normal mitotic division
(Fig. 17). Univalents were found lagging at the equatorial region
in three of 27 pairs of daughter cells recorded. In each pair one
laggard occurred in both daughter cells. Misdivision of lagging uni-

■

valents was also noted in a few cells.

During telophase II, daughter cells were observed to have a
group of chromosomes at each pole and from zero to four univalents
lagging in the equatorial region (Fig. 18). No laggards of any sort
occurred in 54*BO per cent of 177 cells examined. Lagging univalents
either l) misdivided at the plate (Fig. 18), 2) remained intact in the
cytoplasm but were excluded from the main polar group (Fig. 19),
probably to form micronuclei later, or 3) were included with the polar
groups. Misdivision of a lagging univalent was observed in six pairs of
daughter cells and inferred in another seven from pairs of fragments
that were noted (Fig. 19). This indicates that misdivision occurred in
a total of 7*35 per cent of telophase II pairs of daughter cells. Mis¬
division at anaphase I - telophase I was inferred from a fragment noted
in each cell of seven (3.95 per cent) of cells. Five pairs were recorded
with one to three fragments. In all of these observations, made on 177
.pairs of daughter cells, the fragments were the size of univalent
chromosome arms; hence, they probably originated from misdivision of
lagging univalents at first and second division.

At telophase II a microcyte was seen to be associated with 5.09
per cent of the pairs of daughter cells (Fig. 20). The diad within the
microcyte divided equationallv and the two halves moved to opposite ends
of the minute cell, apparently following the same procedure as diads in
normal cells. It was noted, however, that throughout second division the
process lagged behind that in the main cells in passing through the stages
of division. For example, in Fig. 20 the two main daughter cells are at
late telophase while the microcyte is at the anaphase stage.

• .

. .

■

■ ■

,.'s tfg&fcvhb ^.y-'to^y

■

. ■

■

hcj hij hrj *ij *Trj

Fig.15. Interphase cell illustrating attached microcyte with nucleus
formed from single diad.

Fig.16. Metaphase II with 10 diads in one and 11 in the other daughter
cell.

ig.17. Anaphase II daughter cell with 11-12 distribution.
ig.18. Anaphase II - telophase II with misdividing daughter-univalent
in each daughter cell.

ig.19 Telophase II with lagging fragments and daughter-univalents.

ig.20 Telophase II with divided diad in microcyte.

ig.21. Tetrad with associated twin microcytes.

. •

. -

Tetrads were found to have from zero to nine micronuclei and
an average of 1.81 per tetrad in 1346 that -were recorded. A minute
microspore containing one micronucleus was attached to 2.30 per cent of
the tetrads. Presumably these were formed from univalents and fragments
that had been isolated in the cytoplasm at second division.

At telophase II 54.80 per cent of the pairs of daughter cells
had no lagging univalents or fragments. Assuming that laggards at this

stage were the source of micronuclei in the tetrads, a similar or perhaps
even greater proportion (because of laggards eventually included in the
main nuclei) of tetrads should have occurred without micronuclei. Un¬
expectedly, however, only 29*72 per cent of the tetrads had no micronuclei.

Microcytes initially formed from anaphase I peripheral diads
were associated with 2.16 per cent of the tetrads. They now appeared
as single minute cells, each with two nuclei, or as twin microspores,
each with a micronucleus. These microcytes were enveloped together with
the main group of four microspores by a common sheath (Fig. 21). The
lower proportion of these microcytes observed at the tetrad stage (2.16 per
cent) when compared with their proportions at telophase II (5.09 per cent)
and anaphase I (8.06 per cent) can be explained by their separation and
loss from the tetrad envelope during preparation of the slide, in spite
of the care taken to avoid this.

Restitution at second division was indicated in four cases by
the association of one large microspore with two of normal size.

Viability of Pollen from Triploids
To obtain an estimate of the proportion of good pollen, nearly

mature anthers from secondary tillers of one triploid plant were treated
with an iodine solution. Grains were classed as good if they appeared
large, well filled with starch, unshrunken, and comparable to those
observed in a diploid plant (fig. 22). The data in Table XIV show that
there was considerable variability between anthers in the proportion of
good pollen. The range was from zero to 21.3 per cent, the average
being 5*5 per cent. Diploid plants grown under comparable conditions
produced 98.7 per cent good pollen, figs. 23 and 24 give an indication
of the types of grains that were formed in two anthers from tripioids.

TABLE XIV

Percentage of good pollen from
triploid and diploid plants of Gateway

Source

Total no.
of grains

No. of
good grains

% good
grains

Diploid

5200

5132

98.7

Triploid

Anther 1

1013

0.0

n 2

672

0.2

" 3

1007

4*6

" 4

1010

4.0

" 5

561

2.9

” 6

1003

4.9

1108

226

21.2

Total 6374

349 Av

. 5.5

Fertility of Tripioids

The fertility of all well-developed heads of open-pollinated

hrj >xj

ig.22. Pollen 98 per cent good from diploid plant.
ig.23. Pollen 2.54 per cent good from anther of
triploid.

Fig.24. Pollen zero per cent good from anther of
triploid.

triploid plants was calculated by expressing the number of seeds as a
percentage' of the total number of florets. Data on the fertility of
cytologically identified Gateway and hybrid triploids, Gateway triploids
pollinated with diploid Gateway, and Gateway control plants are
presented in Table XV. All plants were grown in field plots.

TABLE XV

Fertility of triploids and diploids

Source

No. of
florets

No. of
seeds

fertility

Open-pollinated
Gateway triploids

1413

4.2

Gateway 3x x 2x

142

6.3

Ope n-pollinat ed
hybrid triploids

1024

119

11.6

Cpen-pollinat ed
Gateway diploids

1531

1470

96.0

The average fertility of 10 Gateway triploid plants was 4*2 per cent
and of three hybrid triploids 11.6 per cent. Although these values are
not directly comparable, since the triploids from the two sources were
grown in different years, the hybrid plants on the average were more
fertile. Ten diploid Gateway plants taken at random from the same plots
as the triploids had an average fertility of 96.0 per cent. The data in
Table XV indicate little difference in seed set between Gateway triploids
that were open-pollinated and those hand-pollinated with normal diploid
Gateway.

Data on the viability of seeds from triploid plants are
presented in Table XVI. Approximately 54 per cent of the seeds from

the Gateway and 45 per cent of those from the hybrid triploids germinated.
Although the germinability of the seed from the Gateway triploids was
higher, the ultimate survival of the hybrid seedlings was about 50 per
cent greater.

TABLE XVI

Viability of seed from triploids

Adult plants

Source

No. of

seeds planted

Germination

No • %

No.

% of

germinated

seeds

Gateway triploids

32 54.2

62.5

Hybrid triploids

119

54 45.4

92.6

Progeny of Triploids

The number of the various chromosome types recovered among
the progeny of Gateway and hybrid triploids is given in Table XVII.

TABLE XVII

Chromosome constitution of progeny of triploids

Source

No. of

No.

of adult plants

of seed

seeds sown

Total

2 n

2 n+l

2 n+2

2 n+3

? n

Other

Unknown

Gateway

triploids

Hybrid

triploids

119

2 n*f(2)

2 n+l+f

2 n+2+f

Total

200

Approximately 38 per cent of the seeds that were sown produced mature
plants• Of these, 24 per cent were normal diploids, and approximately
51 per cent were primary trisomics. Three plants had three extra
chromosomes; this was the maximum number of extra chromosomes found
among the aneuploids. Four had a fragment in addition to one or two
extra chromosomes. One plant was identified as triploid. In general,
aneuploid plants with more than one extra chromosome were dwarfed,
lacked vigor, had few tillers, and all but three of 12 plants in this
catagory were completely self-sterile. Characteristics of 2n + 1 plants
are described under the second part of this study.

DISCUSSION

The apparent difficulty in producing triploids of common
barley experimentally is indicated by the paucity of reported attempts
and the failure noted in the present study. Undoubtedly, the method of
producing triploids of barley by intercrossing tetraploids and diploids
has been unsuccessfully attempted numerous times and, consequently,
has not been reported. Although Tsuchiya ( 64 ) was successful in
obtaining a triploid by this method, his triploid cannot be considered
a true autotriploid of common barley since the diploid involved was a
closely related vvild species. Horde urn spontaneum . His success with this
particular cross suggests that combinations between widely divergent,
unrelated stocks of tetraploids and diploids of common barley are more
likely to be fruitful as a source of triploids. Based on the few known
attempts the conclusion can be drawn that crosses between tetraploids

■

• ' ■ 1

o . .

and diploids of common barley are highly incompatible.

Barley triploids have been reported to occur spontaneously as
members of-twin seedlings (37)* The present study has provided evidence
that they occur spontaneously in another manner. Of three triploids
found in a hybrid F^ population derived from colchicine treated F-j_ plants,
one -was noted among the progeny of a mother plant that also produced
diploids and tetraploids. Presumably, it originated from the union of
a haploid and a diploid gamete produced by the treated mother plant.

The other two triploids were descendents of treated F^ plants which
otherwise produced only normal diploid offspring, as indicated by their
normal fertility. Consequently, the mother plants of these presumably
had produced only haploid gametes. Apparently, therefore, either 2x
pollen from other nearby treated plants participated in fertilization
or the two triploids were of spontaneous origin; that is, they were not
the result of colchicine treatment. The latter supposition is more
likely since it has been shown that 2x pollen usually does not function
in 2x X 4x crosses (10, 12, 19, 14, 16). Further evidence to support
the hypothesis of the spontaneous occurrence of triploids in this manner
was provided by an additional 13 that were found in a nursery of
approximately 4500 progeny head-rows of treated and untreated Gateway
barley. Since three of these were found among the progeny of untreated
mother plants, it is assumed that these and probably a portion of those
from treated plants were of spontaneous origin. The sibs of all triploid
plants but one were fully fertile and, therefore, presumably diploid. In
the one exception two triploids were found in the same family.

It is likely that none of these spontaneous triploids were
derived from twin embryo seeds, since according to Muntzing (37) when

. ■

•. .. .. ....

. ‘ y

one member is triploid the other is diploid and, consequently, completely
fertile. All spikes produced by the triploid plants described in this
study were highly sterile; hence, none could have been members of
diploid-triploid twins. It is probable that they resulted from the
fertilization of an unreduced by a reduced gamete produced by otherwise
normal diploid plants. Cytological studies of several diploid species
have shown that unreduced gametes are occasionally formed (2, 8 ,

35 9 42, 66). The writer has observed such gametes also in cytological
studies of diploid barley. The evidence from the present and other
studies further indicates that the triploid plants, described as being
of spontaneous origin in this study, probably resulted from the
fertilization of diploid eggs with haploid pollen produced by normal
diploid plants; diploid pollen most probably was not involved since, as
stated above, studies have shown that it rarely functions in the
fertilization of diploid plants.

Undoubtedly, triploid barley plants of spontaneous origin have
been rarely observed in natural populations because of their close
resemblance to normal diploids and the consequent difficulty in
detecting them. 'The triploids described in this study were noted in
field plots only because of their sterility which at the time of
flowering was indicated by florets which remained open for an abnormally
long period and by a very low set of seed on mature spikes.

A higher proportion of trivalents and, consequently, lower
proportion of univalents was found in the Gateway triploids than
Tsuchiya noted in his triploid ( 64 )• These differences can be attributed
to incomplete homology between the chromosome sets of the parents of

'■ "1 ;

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. . J . . V v.'. ’■ jdt ■ ‘ ft; ' .. I

. . ,

■.

■

Tsuchiya’s triploid, common barley and Hordeum spontaneum .

Only three quadrivalents were noted in 1092 metaphase I cells
of the Gateway triploids. This substantiates evidence obtained from a
haploid ( 62 ) and a hypotriploid ( 63 ) to indicate that very little
reduplication of chromatin occurs within the basic set of chromosomes
of common barley.

Univalents observed at metaphase I in the Gateway barley
triploids were divided into two groups: l) those located off the
equatorial plate and 2) those found on the plate. By means of suitable
statistical treatment of the data it was shown that the univalents
positioned off the plate were distributed on opposite sides of it at
random. These results agree with those reported by Myers (39) who
studied univalent behavior in triploid Lolium perenne . As might be
expected the relative proportions of these two classes of univalents
has been found to vary among different triploids (36, 39* 64 ). However,
of more significance is the behavior of each group at late metaphase I
and at anaphase I. In his study Myers (36) noted that an average of 1.33
laggards per cell occurred at anaphase I, as compared with a total
average of only 0.93 univalents per cell at metaphase I. He attributed
this excess at anaphase to "improper" disjunction of trivalents. Although
Tsuchiya ( 64 ) noted a much lower average proportion of laggards at
anaphase I than of average total univalents at metaphase I (I .46 per cell
as compared with 2.33)* he found that there was a greater proportion of
anaphase I laggards than could be accounted for by the proportion of
univalents located on the plate at metaphase I. He assumed that only
univalents located on the metaphase I plate lagged and divided
equationally at anaphase I and concluded that the excess of anaphase I

■

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laggards originated from "improper" disjunction of trivalents. The

results from the present study indicate that trivalents were not a

source of lagging univalents at anaphase I in Gateway barley triploids.

An average of 0.66 univalents per cell were found on the plate at

metaphase I and 0.80 laggards per cell at anaphase I. The difference

between these values is considerably less than between the corresponding

values for Tsuchiya's triploid. A X test of observed frequencies of
anaphase I cells with zero to five laggards and the expected frequencies
based on the proportion of metaphase I cells with corresponding numbers
of univalents on the plate indicated that there was close agreement for
all but the two classes with four and five laggards (Table X). The
number of cells observed in these two classes was too small (four per
cent of the total) to permit a valid test. The average for the remaining
four classes, having from zero to three univalents on the metaphase
plate, and that for the corresponding classes of anaphase laggards was
0.6 k univalents and 0.69 laggards per cell, respectively. Since these
two values are in close agreement, the conclusions can be drawn that
only those univalents located on the plate at metaphase I lagged and
divided equationally at anaphase I and that members of trivalent
associations were not an additional source of anaphase laggards.

The assumption that the extra set of chromosomes in triploids
is distributed to the poles at random during meiosis has been inferred
in several studies of triploids but has been adequately tested in only
two (37, 51> 52). In the present study the statistical analysis of the
data obtained from a relatively large number of cells indicated that at

■

. .. .......

. If . ~ • • , : . ' . •' «, 1 .

■ ' ■ > . . .

anaphase I the chromosomes were distributed to the poles in a binomial
frequency in all classes of cells, with and without lagging univalents.
(Tables XI and XII). These results agree with Myers*(39) conclusions,
based on a study of triploid Lolium perenne , that n the distributions at
anaphase I also were consistent with the hypothesis of chance position
of unoriented metaphase I univalents and random assortment of the extra
chromosomes of the trivalents." That these findings do not have general
application to all triploids has been shown by Satina and Blakeslee (51)
in their detailed study of triploid Datura stramonium . They noted a
statistically significant divergence of certain distribution classes
from that expected according to random assortment at anaphase I. They
concluded that ”it seems probable that the divergence of the assortments
at the I division in P.M.C. from calculated values is of general occurrence
and is to be attributed to the nature of the chromosomes and the mechanisms
involved in their movements at division.”

The results from the present and from previous investigations
of triploids emphasize the caution to be taken in forming broad, general
conclusions from a single study and also indicate the need for further
intensive, detailed statistical analysis of chromosome behavior in
triploids of other species.

Although the precise behavior of the chromosomes was difficult
to trace at second division in the material available for this study,
a general pattern could be perceived. At metaphase II - anaphase II the
diads formed an equatorial plate and divided equationally. Univalents
derived from division of anaphase I laggards were either positioned on
the plate or lagged in the cytoplasm at metaphase II. Subsequently

their behavior at anaphase II - telophase II followed one of three
courses: 1) they were included with the polar groups; 2) they remained
lagging intact in the cytoplasm, probably to form micronuclei later;
or 3) they misdivided at the equatorial region in a small proportion of
cells. As a result of the division of lagging chromosomes at anaphase I
- telophase I and the subsequent lagging of daughter-univalents and
fragments at second division, tetrads with an average of 1.81 micronuclei
were formed. No micronuclei occurred in approximately 30 per cent of
the tetrads. This is an unexpectedly low frequency when compared with 55
per cent of the pairs of telophase II daughter cells that contained no
micronuclei.

Microcytes possessing one or two chromosomes have been observed
associated with normal cells at meiosis in a variety of plants, including
triploids of Datura (7), liliurn (13), hyacinths (18), Zea (33) and
Hordeum ( 64 ). In the present study it was possible to follow the
behavior of microcytes through all stages of both meiotic divisions. They
originated at anaphase of first division from one or, less often, two
univalents that were positioned on the periphery of the equatorial region
or of a polar group of chromosomes. These univalents showed the typical
anaphase ’split* but the halves did not separate. At telophase - interphase
each peripheral diad formed a minute cell, although in rare instances two
were seen together in a single microcyte. Each minute cell was attached
to the microspore mother cell (Fig. 15). At second division the
chromosome(s) in each microcyte divided equationally, and the resulting
univalent-halves moved to opposite ends of the microcyte. At the
completion of second division each microcyte had developed into twin minute

„

microspores that were attached to the main group of four (Fig, 21),

These observations are similar to those reported in hyacinths (18),

Lilium (13) and Triticum (23)* They indicate that a minute cell with
a single chromosome is capable of independently undergoing division,
exhibiting spindle activity and polarity during the process, and they
add to the evidence for the autonomous nature of individual chromosomes
during meiosis.

Although the distribution of the extra set of chromosomes has
been shown to be random or to approach randomness in a number of different
triploids, in none has the frequency of functional eggs and pollen been
found to correspond to the expected. Usually gametes with chromosome
numbers approaching the haploid complement of the species were found to
function most frequently. 'Those with intermediate numbers and numbers
near the diploid complement functioned infrequently, rarely, or not at
all. Furthermore, it has also been noted that there is usually a more
pronounced selection against aneuploid gametes on the male than on the
female side. The results of the study on triploid Gateway barley agree
with these general observations in other species. On the basis of random
assortment of the chromosomes at anaphase I, plants with 18 to 24
chromosomes would be expected most frequently. It was found, however,
that approximately 75 per cent of the progeny from open-pollinated
triploid plants were diploids (14 chromosomes) and primary trisomics
(15 chromosomes). Apparently, gametes vjith more than one extra chromosome
functioned infrequently, and most probably these were female. The low
frequency of progeny with more than one extra chromosome may be attributed
to the following factors, assuming that chromosome distribution at

' , ,

■

. . . ' .

macrosporogenesis 'was also random: l) Gametes with more than one extra
chromosome were probably less viable than those with the haploid number
of seven or with eight. 2) Male gametes with more than one extra
chromosome were less viable than female gametes with the same number
of chromosomes. 3) Male gametes with extra chromosomes probably failed
to function in fertilization because of certation. 4) Aneuploid embryos
with more than 15 chromosomes were probably less viable than diploid

and trisomic embryos, as suggested by the low fertility of the triploids.

5) Reduced seed germination and seedling lethality probably were
further effects of 4)*

A comparison of the fertility among the triploids from the
two sources reported in this study and Tsuchiya's triploid would seem
to indicate that fertility of triploids is influenced by the degree
of heterozygosity. The average fertility of the triploid plants
obtained from a highly homozygous stock of the variety Gateway was four
per cent, of those derived from highly heterozygous, intervarietal
hybrids about 12 per cent and of Tsuchiya’s interspecific hybrid triploid,
produced from the cross between tetraploid common barley and the closely
related wild diploid species, Hordeum spontaneum , 19 per cent. Thus,
the triploid plants from the most homozygous stock were lowest in fertility,
while the triploid from the interspecific hybrid was highest, despite
the higher proportion of univalents found at meiosis in the latter.

. u\OC' ■

■

- - - . ■ - - ■■■.

j: • . v.\ •• s: ■ o';,i

SUMMARY

1. Three triploid plants were found in an F£ population of
common barley derived from colchicine treated intervarietal hybrids.

An additional 13 were found in a large population of the variety Gateway.
Triploids occurred spontaneously in Gateway with a frequency of one in
approximately 6000 plants; the origin of these was attributed to the
fertilization of unreduced female gametes with reduced pollen produced
by diploid plants.

2. Morphologically, adult triploid plants were indistinguishable
from diploids.

3. An attempt to produce triploids by pollinating tetraploid
Gateway with the related diploid was unsuccessful.

4. At meiosis in the Gateway triploids an average of 5«22
trivalents and 1.78 bivalents occurred. All types of trivalents that
are. possible from pairing between three homologous chromosomes were
observed. In addition, three quadravalents were noted in 1091 cells
examined.

5. It was statistically determined that univalents lying off
the equatorial plate at metaphase I were distributed on opposite sides
of the plate at random.

6. 1? analysis of the data indicated that univalents located
on the plate at the completion of metaphase I lagged and divided
equationally at anaphase I. Univalents off the plate did not divide but
were included with the nearest polar group at anaphase I.

7. The distribution of the chromosomes to the poles at
anaphase I was in a binomial frequency and, therefore, in accordance

■

with the hypothesis of random assortment.

8. Misdivision of lagging univalents occurred in 1.90 per
cent of telophase I cells.

9. At second division diads divided equationally. Univalents
derived from equational division of lagging chromosomes at anaphase I
either were included with the main anaphase II polar groupings or
lagged in the cytoplasm. Misdivision of univalents at telophase II

was inferred in 3*95 per cent of the pairs of daughter cells.

10. As a result of chromosome lagging and misdivision at first
and second meiotic divisions, about 70 per cent of the tetrads had one

or more micronuclei.

11. Minute twin microcytes were associated with 2.16 per cent
of the tetrads (Fig. 21). Each set originated from a lagging univalent

located on the periphery of the cell at anaphase I where it formed a
microcyte at telophase I - interphase I (Fig. 13). During second
division the microcyte behaved as an independent cell; the enclosed
univalent divided equationally to form daughter cells, each with a
chromatid. (Figs. 20, 21).

12. Pollen from one Gateway triploid plant averaged 5*5 per cent

good.

13. The fertility of open-pollinated Gateway and hybrid
triploids was 4*2 and 11.6 per cent, respectively (Table XV).

14. Approximately 54 per cent of the seed from the Gateway
triploids and 45 per cent from the hybrid triploids germinated.

15. Of a total of 75 offspring obtained from the triploids,

24 per cent were diploid, 51 per cent were trisomic, and one plant was
triploid. None of the aneuplcid plants had more than three extra chromosomes.

'idD

PART II: TRISOMICS

REVISE OB’ LITERATURE

The first trisomic plants -were described in Datura in 1919
by Blakeslee and Avery (2), In 1920 Blakeslee et al*(5) reported that
12 morphologically distinct trisomic types were found, corresponding to
the haploid chromosome number of Datura , By means of the modified ratio
technique Blakeslee and Farnham (4) identified the gene 'white 1 with the
trisomic type 'Poinsettia'. Subsequently, genes were identified with
each of the 12 trisomic types. Since these early reports on Datura ,
trisomics have been obtained and studied in Nicotiana (12, 16), tomato
(17, IS, 19, 27), corn (21, 22), rye (30), and more recently in common
barley (25, 28, 31)*

McClintock and Hill (22) used trisomics to associate the gene
X with the smallest chromosome pair of corn and also reported the ident¬
ification of trisomics with five additional linkage groups. Lesley
(17, 18, 19, 20) first obtained 11 of the 12 primary trisomics in tomato
and identified certain of these with specific linkage groups. More recently
Rick and Barton (27), using different material, obtained the complete
series of 12 primary tomato trisomics and cytogenetically identified six
with the corresponding linkage groups.

Trisomic plants have been obtained from several sources.
Occasionally they have occurred in the progeny of normal diploids and
presumed to have resulted from nonconjunction or nondisjunction at meiosis
(7, 12, 30). A further source has been from the progeny of plants
heterozygous for an interchange. Burnham et al. (ll), and Ramage (25)
attributed the origin of these to a 3 ; 1 disjunction from a translocation

- i U . t

. • • . . V.

■ • . - •• * --•"

... •

complex of four chromosomes and subsequent formation of gametes with one
extra chromosome. The most prolific source, however, has been from the
progeny of triploids (6, 8, 13, 14, 16, 18, 21, 23, 24, 27, 31).

In most species the presence of an extra chromosome changes the
phenotype of the plant. Usually a group of characters specific for each
chromosome is altered. Thus, in Datura the 12 primary trisomic types
were phenotypically distinct (l), as were those of Nicotiana sylvestris
(l6) and tomato (27)* On the other hand, none of the chromosomes in
corn produced distinctive morphological changes when in triplicate other
than decreased size and vigor (21). Characters that have been observed
to be modified by the presence of an additional chromosome include
growth habit; plant height; shape, size, color and position of the leaf;
thickness and stiffness of the stems; and enlargment or decrease in size
of the floral parts and fruits. In addition, trisomic plants generally
have been noted to be less vigorous than diploids and completely to
partially sterile, particularly on the male side (16, 18, 21, 25 , 27, 31)*

On the basis of random chromosome distribution to opposite
poles at meiosis, gametes with the haploid number and with an extra
chromosome should be produced by trisomics in equal frequencies.
Furthermore, if both male and female gametes are fully functional, equal
proportions of diploid and trisomic progeny should be produced. Actual
transmission of the additional chromosome is, however, considerably less
than expected and varies with the trisome involved. In Datura (3)
transmission through the ovules varied from about three to 33 per cent;
in tomato (27) from less than one per cent to about 25 per cent, and in
corn from 22 to 52 per cent (15)* The frequency of transmission through

the pollen has been found to be considerably less than through the eggs.
Under favorable conditions only five of the 12 primary trisomics of Datura
were transmitted through male gametes (9). In Nicotiana sylvestris
transmission through pollen varied from zero to 34 per cent, and in
eight of 11 trisomics it was less than 10 per cent (16). In corn McClintock
and Hill (22) found that approximately 1.4 per cent of the progeny of one
trisome were trisomic from the cross 2n I 2n + 1. Rhoades ( 26 ) found no
trisomics in a population of 1845 plants of a similar cross involving a
different trisome of corn.

In common barley Smith (28) found trisomics in a diploid stock,
the origin of which he attributed to a 6:8 chromosome segregation at
meiosis. Later, Ramage (25) noted that an interchange was carried by the
same stock and concluded that Smith’s trisomics likely were the result
of a 3:1 separation from a ring of four chromosomes. Although the
trisomics from this material were shorter than normal, they were
vigorous and had no distinguishing characteristics. In 1952 Tsuchiya (31)
recovered six trisomic plants among the progeny of a hypotriploid plant.

They differed from each other and from diploids in several characteristics,
including plant height; stem thickness; number of tillers; length, width
and color of leaves; shape of heads; habit of growth; time of maturity;
fertility; and cytological behavior. The most striking characteristic
among the trisomics was their fertility, which varied from zero to 92
per cent under self-fertilization.

In 1955 Ramage (25) was the first to report on the morphological
and cytogenetical identification of barley trisomics. He isolated primary
and secondary trisomics from interchanges involving each of the seven

i t • a! •

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chromosomes of the variety Mars. The morphological descriptions of

the trisomic types found to be genetically associated with specific

chromosomes were given as follows:

■Trisome of Chromosome a 1 .- This type was
weak, dwarf and highly sterile.

Trisome of Chromosome b .- This type had
narrow, dark-green leaves.

Trisomes from Interchange c - d .- One type was
dwarf with slender stems and short, narrow leaves;
it was completely self-sterile; most spikes did
not reach the heading stage. The second type from
this interchange was later in maturity than normal
but otherwise was indistinguishable from diploid sibs.

Trisomes from Interchange e - f .- One type
was dwarf with short, wide leaves, particularly the
flag leaves. The other type was readily distinguished
by the long, narrow, light-green, drooping leaves.

Trisome of Chromosome g .- This type was
not readily distinguished from diploid
sibs, although it produced fewer tillers.

Burnham and Hagberg (10) have summarized the results obtained
by Eamage and by several other workers ?dio have utilized trisomics and
translocation stocks to determine the association of barley linkage
groups with their respective chromosomes. The evidence establishes the
independence of all linkage groups except III, VII, and V, on the one
hand, and II and V on the other. No genes have been located on
chromosome jg; Burnham and Hagberg suggested the possibility that a and d
also have no known genetic markers. According to their summerization, the

following associations appear probable at the present time:

Chromosome f b c e a g d

Linkage Group I III VI IV II? - V?

VII

The seven barley chromosomes have been designated
temporarily by the small letters a to £ by Burnham et al. (ll).

«f

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MATERIALS AND METHODS

Primary trisomics described in this part of the study were
obtained from the progenies of the hybrid and Gateway triploids discussed
in the first part. Each original trisomic plant was cytologically
identified as such and affixed with a 1 T* number. All trisomic
descendents of each original trisomic plant retained this number. The
morphological descriptions are based on data and notes taken on the
original trisomics and their trisomic progenies grown in the field in
two different years. Measurements on maximum leaf width and length
were taken on the three uppermost leaves of the original trisomic plants
and diploid sibs. Data on spike density were taken on a minimum of
19 spikes collected from six or more plants of each trisomic type and
from the diploid. Data on the number of days required to head are based
on a minimum of six plants, while the average values for plant height were
determined from not less than five plants. All measurements were made on
plants grown in the field in 1955 > except of leaf width and length,
which were obtained from plants grovjn in the greenhouse.

The varieties Nigrinudum I and Golsess V were used as tester
stocks to determine the association of Gateway trisomic type T39 with
its corresponding linkage group. These two tester stocks together possess
at least one contrasting marker gene for the corresponding locus in
Gateway for each of the linkage groups. The characters used, their
symbols, the linkage group to which each has been assigned (29) and the
genotype of Gateway are summarized in Table I. The two genetic stocks
were used as pollen parents in crosses with trisomic plants. Seeds from

trisomic plants were space-planted to facilitate classification
for seedling characters. Individual F^ plants were classified for
each segregating character. Tests for association were made by
analysis of populations for disomic and trisomic ratios.

TABLE I

Linkage groups and marker genes involved in
tests for association with Gateway trisomic T39

Linkage group

Character

Gene

symbol

Genotype
of Gateway

Two-row vs. six-row spike

V, v

Black vs. white pericarp
and lemma

B, b

III

Covered vs. naked caryopsis

N, n

Hooded vs. awned spike

K, k

Blue vs. white aleurone

Bl, bl

blbl

Rough vs. smooth awns

R, r

Normal vs. albino seedlings

A n> a n

VII

Normal vs. chlorina seedlings

F , f
c ; c

OBSERVATIONS AND RESULTS

Morphological Characteristics of Primary Trisomics

Primary trisomic plants obtained from the progeny of the
triploids differed from one another and from diploid sibs in characteristics
such as rate of growth; relative vigor; height; length, width and

■

. .

.) -J

color of leaves; degree of tillering; and length and density of the spike.

Although a few of the trisomic types derived from the hybrid triploids

appeared to have certain distinct characteristics, it was difficult or

impossible to distinguish these trisomics morphologically from one another

and in some instances from the diploids because of the high degree of

heterozygosity present in the original stocks. One type, however, differed

conspicuously from all others and from the diploids by having extremely

long, narrow, drooping leaves with enlarged auricles and ligules. This

also was the only type that could be distinguished in the seedling stage.

A similar type occurred among the trisomic progeny of the Gateway triploids*

Since the Gateway trisomics were derived from a pure line stock,

morphological differences among them could be attributed to the effects of

specific chromosomes when present in triplicate.

On the basis of the data on some measurable characteristics

given in Table II and on additional visual differentiating characteristics,

four Gateway trisomic types could be readily distinguished from one

another and from diploids. Compared with diploids, all trisomic types

were shorter, later in heading, had fewer tillers and were considerably

lower in fertility. Specific differences between the four types and

between these and the diploids were as follows 2

Type T31(Fig, l) .- Leaves darker green,

broader and more erect than diploid; neck distinctly

coiled (Fig. 2); head relatively short and dense

when compared with diploid and other trisomics;

awns appressed rather than spreading;

tallest of the four trisomics.

Type T39(Fig. 3 ) .-Spike shorter but more lax
than diploid and tended to be tapered from base
toward apex, rather than oblong in shape; leaves
shorter and narrower than normal and rolled under
at the margins, particularly toward the apex;
compared with other trisomics, this type had the

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Comparative data on morphological characteristics
of Gateway trisomic types

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longest spike, was most prolific in stooling and had
the greatest fertility.

Type T46 .- Leaves extremely long, narrow and
drooping with relatively large auricles and
ligules; head emerged from the side of flag leaf.

Type T51 (Fi g- /|)-~ Leaves very broad relative to
length, particularly the flag leaf, dark blue-green
in color, erect, and base tended to clasp the thick
stem; florets were small and flaccid at the heading
stage with supernumery organs on the upper ones; at
emergence from sheath the awns projected in all
directions giving spike a ragged appearance.

The original trisomic type T46 from the Gateway stock was lost because

of high sterility. However, a very similar type (previously referred to),

probably trisomic for the same chromosome, was recovered from the

hybrid material; it reproduced readily under open-pollination.

When all of the adult characteristics of each Gateway trisomic

type were taken into consideration, it was relatively easy to distinguish

them from each other and from the diploid under field conditions. In the

greenhouse certain of the differentiating features tended to be modified

or absent. For example, T51 did not show the extremely broad, dark-green

leaves and thick stems in the greenhouse, and the development of the

head and florets was more normal, facilitating emasculation and hand-

pollination.

Fertility of Trisomics

The fertility among 14 original trisomic plants obtained from
the progeny of the hybrid triploids ranged from about 20 to 67 per cent
and averaged 46 per cent under open-pollination in the greenhouse. The
average fertility of nine diploid sibs was about 92 per cent. Twelve
additional trisomics obtained from the same source but grown in the field
had a fertility from zero to approximately 92 per cent, with an average of
about 52 when open-pollinated. Two of the 26 trisomic plants were

■

\ DO'

'i .. ... 3

ig.l. Trisomic type T31 and diploid Gateway.
ig.2. Trisomic type T31 (left)showing coiled neck and
diploid Gateway (right).

Fig. 3 . Trisomic type T39 and diploid Gateway.

Fig. 4 . Trisomic type T51 diploid Gateway.

completely self-sterile. The self-fertility of three morphologically
distinct Gateway trisomic types and the diploid when grown in the
field is given in Table III. Type T31 had the lowest fertility with
a seed set of about 17 per cent, while 'Type T39 was highest with about
50 per cent.

TABLE III

Fertility of open-pollinated Gateway trisomics
and the diploid

Trisomic

type

No. of
plants

No. of
florets

No. of
seeds

fertility

T31

1014

170

16.8

T39

1575

789

30.1

T51

246

_ 2 i

37.0

2835

1050

Av. 37.0

Diploid

663

637

96.1

Transmission of Trisomics

Data on the frequency of trisomic plants among progenies of
open-pollinated trisomics are shown in Table IV. Trisomic plants were
distinguished from diploids in the hybrid progenies by lack of vigor,
short growth, lateness, sterility and certain morphological characteristics
that were known from previous experience to differentiate them. In a few
instances trisomic plants were cytologically verified. The Gateway
trisomics were readily identified from diploid sibs by the morphological
characteristics already described. The frequency of trisomic plants

; '■ .LX ;.'j J' foulS xh

. . ■ ■ ■ .

- . i 1 -- - ■ ' •

L • ■ ■

• ' •

•.

.1 '■ • •.- . ■ ; - - "■ 1 • •

TABLE IV

Frequencies of transmission of
trisomic plants in progenies of open-pollinated trisomics

Trisomic
. type

Total no. of

2 n *• 1 and 2 n

No. of

2 n + 1

Per cent
2 n -f 1

Hybrids

T4-2

22.9

T5-1

17.9

T6-2

17.6

T10-2

21.1

112-1

25.5

215-1

25.0

T16-1

22.2

T 21

31.1

T22

25.0

T23

33.3

T26

36.7

127

20.0

T28

23.5

T29

16.7

130

26.9

562

137

Av. 24*4

Gateway

131

24.6

139

26.4

T51

21.4

175

Av. 24-6

among the progenies of open-pollinated hybrid types ranged from 16.7
to 36.7 per cent, averaging 24.4. The frequencies of transmission among
the progenies of the three morphologically identified Gate-way types
T31, T39 and T51 were 24.6, 26.4 and 21.4 per cent, respectively.

Limited data were obtained on the frequency of transmission
of the extra chromosome through the pollen. Of 237 plants from the cross
2n X 2n +• 1, involving five hybrid trisomic types and four diploid
varieties, only one trisomic plant was cytologically identified (fable V)

TABLE V

Frequencies of transmission of
trisornics in progenies of 2n X 2n + 1

Trisomic

type

Total no. of

2n + 1 and 2n

No. of

2n + l

% of
2n + 1

T12-1

0.0

T15-1

1.5

T21

0.0

T22

0.0

T27

-12

0.0

237

Av. 0.4

Cytogenetic Identification of Gateway
Trisomic T39

Tests were completed for the cytogenetic identification of
Gateway trisomic type T39. The observed F 2 segregations for each of
the marker genes were tested for deviations from a 3 si ratio. The T
analysis of the data in Table VI indicates disomic inheritance for

... ...

- *

i. . v.. n

. i 'I r: ,J

, :.: s:

.. o ■. ‘ 1 '.

: . '. i . .

TABLE VI

X 2

analysis of

F 2 populations of crosses between trisomic
T39 and tester stocks for 3*1 disomic segregations

Linkage

group

III

Marker _ Frequency

gene

Observed

Calculated

281.00

89.00

277.50

-22.-5Q

0.04

0.13

370.00

0.17

0.95-0.50

226.00

no-op

274.50

-SLL-5Q

8.57

25LZ1

366.00

34.28

< 0.001

272.00

95.00

275.25

-21*25

0.04

0.12

367.00

0.16

0 . 95 - 0.50

233.00

84.00

237.75

79.25

0.09

0.29

317.00

0.38

0 . 95 - 0.50

98.00

40.00

O O

ITN U-\

. *

<r\ -4 1

1—1

0.29

0.88

138.00

1.17

0 . 30 - 0.20

351.00

103.00

340.50

m-ip.

0.32

0.97

454.00

1.29

0 . 30 - 0.20

a n

297.00

90.00

290.25

96-7,5

1.18

0.54

387.00

0.72

0 . 50 - 0.30

267.00

82.00

261.75

87.25

0.11

0^22

349.00

0.43

0 . 95 - 0.50

VII

all markers except B, b, located in Linkage Group II. The observed

segregation for this factor was then tested for goodness of fit to a

trisomic ratio based on the assumption of nontransmission of n + 1

pollen, 25 per cent transmission of n + 1 female gametes, and

chromosome segregation. On this basis 7/18 of the F 2 are expected

to be homozygous recessive. The X analysis in Table VII shows
a good fit of observed to calculated values, indicating trisomic
inheritance for B, b. 'The analj^sis of the data in Tables VI and VII,
therefore, establishes the association of trisomic type T39 with
Linkage Group II and its independence of the other known groups.

TABLE VII

X analysis of Fp population for 11:7
trisomic segregation of B, b in Linkage
Group II

Marker gene

Frequency

Observed

Calculated

226.00

223.70

0.02

140.00

142.30

0.0 u

366.00

366.30

0.06

P = 0.95 -

0.50

IIoGUSSIGN

Gn the basis of the descriptions of the trisomics of the
variety Mars and their associations with certain chromosomes, as given
by Ramage (25), it is possible to indicate which of the four Gateway
trisomic types likely correspond with specific chromosomes. Trisomic
type T39 of the variety Gateway was genetically identified with Linkage

Group II and shown to be independent of the remaining known groups.
Therefore, according to the probable associations between the chromosomes
and linkage groups indicated by Burnham and Hagberg (10), type T39
should be trisomic for chromosome a* However, the morphological
description given by Ramage for this trisomic does not agree with that
for T39. Fossibly the morphological characteristics expressed by a
certain chromosome when in triplicate vary from variety to variety. For
example, Ramage observed no trisomic type in Mars with a coiled neck
(personal communication), a characteristic that was very distinct and
invariable f° r type T31 of Gateway. Otherwise, the latter type appears
to correspond to Ramage 1 s trisome of either chromosome c or d, on the one
hand, or g on the other; more likely it is the trisome of g, since this
type produced fewer tillers, which was also a characteristic of 131 •

Ramage*s two trisomic types involving chromosomes e and f (the two types
were not specifically identified as to which of these two chromosomes
was associated with each) probably correspond to Gateway trisomic types
T 46 , which had long, drooping leaves, and to T51, which approached
dwarfness under field conditions and had short, broad leaves, particularly
the flag leaves.

In accordance with the findings on trisomics of other species
(3, 15, 16, 27), the frequency of trisomic plants among the progenies of
open-pollinated barley trisomic types was considerably less than the
theoretical 50 per cent, approaching an average of 25 per cent in the present
study. Limited results also indicated that the extra chromosome was
transmitted through the pollen with a frequency of less than one per cent;
therefore, it is probable that transmission of trisomics in barley is

ifl ‘..J la

■j

4 ;

largely, if not entirely, through female gametes, at least for
certain chromosomes. This is also in agreement with the results
reported in Datura (9), Nicotiana (16), and corn (22, 26).

Both Tsuchiya (31) and Ramage (25) found that certain simple
trisomic types obtained from pure varieties of barley were completely
self-sterile. In the present study two of 26 original hybrid and two
of 12 Gateway primary trisomic plants were completely sell-sterile.

Two additional trisomics of Gateway were lost because of almost complete
self-sterility. This evidence indicates that certain of the primary
trisomes of common barley, particularly if established in a pure variety,
are completely self-sterile, or nearly so. They would have to be maintained
by hand-pollination with pollen from diploids of the same variety or, as
suggested by Ramage (25)* maintained as heterozygous stocks, since
trisomics from intervarietal crosses are more highly fertile.

SUMMARY

1. Four morphologically distinct primary trisomic types
of the variety Gateway were identified.

2. The fertility of each of three Gateway trisomic types
under open-pollination was approximately 17* 37 and 50 per cent,
respectively, averaging approximately 37 per cent.

3. The frequency of trisomic plants among progenies of 15
unidentified open-pollinated hybrid trisomic types varied from about
17 to 37 per cent and averaged 2U per cent. In progenies of three
open-pollinated, morphologically distinct Gateway trisomic types approx¬
imately 21, 25 and 26 per cent of the plants were trisomic, respectively.

■ i

4* Transmission of the extra chromosome through the pollen
of five unidentified hybrid trisomics averaged 0.4 per cent in a total
population of 237 plants.

5. One Gateway trisomic type was found to be associated
with Linkage Group II and independent of the other known groups.

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PART II: TRISOMICS

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